Compositions and methods for identifying and modifying carbonaceous compositions

ABSTRACT

This invention generally relates to natural gas and methylotrophic energy generation, bio-generated fuels and microbiology. In alternative embodiments, the invention provides nutrient amendments and microbial compositions, e.g., consortia, that are both specifically optimized to stimulate methanogenesis, or for “methylotrophic” or other conversions. In alternative embodiments, the invention provides methods to develop nutrient amendments and microbial compositions that are both specifically optimized to stimulate methanogenesis in a given reservoir. The invention also provides methods for the evaluation of potentially damaging biomass formation and scale precipitation resulting from the addition of nutrient amendments. In other embodiments, the invention provides methods for simulating biogas in sub-surface conditions using a computational model.

RELATED APPLICATIONS

This application is a national phase patent utility filing under 35 USC§371, for International (PCT) Parent application ser. noPCT.US2011/040742, filed Jun. 16, 2011, which claims benefit of priorityto U.S. Provisional Patent Application Ser. No. (USSN) 61/355,488 filedJun. 16, 2010, and U.S. Ser. No. 61/495,815 filed Jun. 10, 2011. Theaforementioned applications are expressly incorporated by referenceherein in their entirety for all purposes.

FIELD OF THE INVENTION

This invention generally relates to natural gas and methylotrophicenergy generation, bio-generated fuels and microbiology. In alternativeembodiments, the invention provides compositions and methods formethanol-utilizing methanogenesis; or “methylotrophic”-conversion,including, including utilizing methylamines and other methyl-containingintermediates. In alternative embodiments, the invention providesnutrient amendments and microbial compositions that are bothspecifically optimized to stimulate methanogenesis from coal or othersubsurface carbonaceous materials. In alternative embodiments, theinvention provides methods to develop nutrient amendments and microbialcompositions that are both specifically optimized to stimulatemethanogenesis in a given reservoir.

BACKGROUND OF THE INVENTION

The methanogenic degradation of subsurface carbonaceous material is ofsignificant commercial interest for a variety of reasons includingproduction of natural gas (including methane). Methane is a predominantend-product of anaerobic microbially-mediated organic-matterdecomposition following a variety of carbon-pathways and intermediatesteps.

Recent technological advances have enabled characterization of microbialcommunities and the biogeochemical processes that take place in thesubsurface. These processes generally occur under non-ideal conditionsdue to limiting nutrients and sub-optimal microbial community structure.Under normal sub-surface conditions, microbial gas formed in thesenatural “bioreactors” is generated at very slow rates due to limitednutrients and/or other environmental conditions, e.g., suboptimal waterchemistry, pH, salinity and the like.

SUMMARY

In alternative embodiments, the invention provides compositions,bioreactors, reservoirs, products of manufacture, fluids or muds, orsynthetic consortiums (e.g., manufactured groups of organisms)comprising a plurality of microorganism strains, wherein themicroorganism strains comprise:

(a) at least two, three; four, five, six, seven, eight, nine, ten oreleven or all twelve of the microorganism strains of Consort-ABS1; or

(b) a group (or “consortium”) of different microorganism strainscomprising at least two, three, four, five, six, seven, eight, nine,ten, eleven or twelve different microorganism strains, each straincomprising at least one 16S rRNA gene or nucleic acid sequence selectedfrom the group consisting of a nucleic acid having at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequenceidentity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:8, SEQ ID NO: 10,SEQ ID NO: 11 and SEQ ID NO: 12, or

(c) a group (or “consortium”) of different microorganism strainsconsisting of at least two, three, four, five, six, seven, eight, nine,ten, eleven or twelve different microorganism strains, each straincomprising at least one 16S rRNA gene or nucleic acid sequence selectedfrom the group consisting of a nucleic acid having at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequenceidentity to SEQ ID NO: 1. SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:8, SEQ ID NO: 10,SEQ ID NO: 11 and SEQ ID NO: 12,

wherein optionally each member of the group (or “consortium”) ofdifferent microorganism strains is a different microorganism strain, oreach member of the group (or “consortium”) of different microorganismstrains has a different 16S rRNA gene or nucleic acid sequence,

and optionally the at least one 16S rRNA gene or nucleic acid sequencecomprises a subsequence (a portion) of a 16S rRNA sequence thatoptionally includes (comprises) the fifth and sixth variable (V5 and V6)regions of a 16S rRNA gene,

and optionally the reservoir is an in situ subsurface reservoir, asurface reservoir, a synthetic reservoir or an excavated reservoir.

In alternative embodiments of the compositions, bioreactors, reservoirs,products of manufacture, fluids, muds and/or synthetic consortiums:

(i) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all 12 of themicroorganism strains comprise a member of the genus Acetobacterium, amember of the genus Bacteroidetes and/or a member of the genusSpirochaetes; or

(ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the microorganismstrains comprise a member of the genus Acetobacterium, a member of thegenus Bacteroidetes and a member of the genus Spirochetes.

In alternative embodiments, the invention provides methods ofidentifying and/or characterizing one or more microbes in a subsurfacemethanogenic microbial community, or identifying and/or characterizing anutrient composition that is customized for a specific subsurfacemethanogenic microbial community, comprising:

(a) obtaining or providing one or a set of samples from a subsurfacecarbonaceous formation or formations,

wherein optionally the samples comprise a production water, or thesamples are taken from a core, cuttings or outcrop sample

and optionally the subsurface carbonaceous formation or formationscomprises a coal formation, or a peat, or a lignite, or a bituminouscoal, or an anthracite coal, or a volcanic ash, or a lignite or a ligninor lignin-comprising composition, a coal or a coal analogue(s) or aprecursor(s) thereof, heavy oil, asphaltenes, and/or an organic debris;

(b) determining and/or characterizing the microbial composition, or themethanogenic strains (e.g., a phylogenetic analysis) of the sample orsamples,

wherein optionally all or substantially most of the microbes in thesample or samples are characterized or identified,

or optionally all or substantially most of the methanogenic microbes(the methanogens, or methanogenic strains) in the sample or samples arecharacterized or identified; and

(c) (i) identifying and/or characterizing one or more microbes in thesample or samples that are the most (or relatively more) methanogenic,

identifying and/or characterizing one or more microbes in the sample orsamples that are the most abundant methanogens,

identifying and/or characterizing one or more microbes in the sample orsamples whose distributions are (or distribution is) correlated withthat of most other methanogens in the sample, or whose distributions are(or distribution is) correlated with the highest level of methanogenesisin the sample, and/or

identifying unfavorable endemic microbes or conditions showing negativecorrelation to biogas formation; or

(ii) applying to the sample or samples a plurality of (a variety of)nutrients mixes and determining a consensus and/or optimal (optimal formethanogenesis) nutrient mix,

wherein optionally the consensus and/or optimal nutrient mix is at leastinitially based upon known requirements of methanogenic microbes, ororganisms associated with methanogenic microbes, and/or fieldobservations of subsurface methanogenic environments,

and optionally the sample or samples comprise a subset of the microbialcomposition of the sample or samples of step (b), or a subset of themethanogenic organisms identified, or characterized in step (b), or aset of methanogenic organisms, identified or characterized in step (c),

and optionally the consensus and/or optimal nutrient mix is alsodesigned to decrease the amount of other (non-methanogenic) bacterialprocesses affecting biogas formation, or to provide an environmentunfavorable to endemic microbes or conditions that show a negativecorrelation to biogas (e.g., methane) formation.

In alternative embodiments, the methods further comprise introducing theconsensus or optimal nutrient mix of 3(c)(ii) to a methanogenicmicrobial community; wherein optionally the methanogenic microbialcommunity is in situ in subsurface methanogenic microbial community.

In alternative embodiments, the microbial composition of step 3(b) isdetermined and or characterized by nucleic acid (e.g., DNA, RNA)sequencing all or a portion of an rRNA gene; or a 16S rRNA gene. Inalternative embodiments, the microbial composition of step 3(b) isdetermined by a chemical, microbiological or any analytical method.

In alternative embodiments, the chemical or microbiological analyticalmethod comprises a fatty acid methyl ester analysis, a membrane lipidanalysis and/or a cultivation-dependent method;

In alternative embodiments, the methanogenic organisms (methanogenicstrains) comprise one or more, members of the Archaea family, or areanaerobic organisms, or are autotrophs or chemoheterotrophs; or themethanogenic organisms comprise one or more members of a genus selectedfrom the group consisting of Methanolobus, Methanobacterium,Methanothermobacter, Methanogenium, Methanogenium, Methanofollis,Methanoculleus, Methanocorpusculum, Methanococcus, Methanocalculus,Methanobrevibacter and Methanosarcina; or the methanogenic organisms(methanogenic strains) comprise or consist of at least one syntheticconsortium of the invention, or one or more members selected from thegroup consisting of:

-   -   Methanolobus bornbayensis    -   Methanolobus taylorii    -   Methanolobus profundi    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschalkii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofollis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeta concilii    -   Methanosaeta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospirillium hungatei    -   Methanothermobacter defluvii (Methanobacterium defluvii)    -   Methanothermobacter thermautotrophicus (Methanobacterium        thermoautoirophicum)    -   Methanothermobacter thermoflexus (Methanobacterium thermoflexum)    -   Methanothermobacter wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii.

In alternative embodiments, the invention provides methods ofdetermining a nutrient composition that is customized or optimal for aspecific subsurface methanogenic microbial community comprising thefollowing steps:

-   -   a. obtaining a sample or a set of samples from one or more        subsurface carbonaceous formation(s) of interest,    -   wherein optionally the subsurface carbonaceous formation or        formations comprises a coal formation, or a peat, or a lignite,        or a bituminous coal, or an anthracite coal, or a coal        analogue(s) or a precursor(s) thereof, a heavy oil, asphaltenes,        and/or an organic debris;    -   b. determining or characterizing the microbial composition of        the methanogenic microbial community of the sample or samples;        and    -   c. growing or culturing one or more enrichment cultures of all        or a subset of tire, microbial composition on a carbonaceous        substrate; a chemical analog, a methanogenic substrate or a        combination thereof,

wherein optionally the enrichment cultures are designed to distinguishdifferent methanogenic pathways, and

-   -   (i) identifying and/or characterizing one or more methanogens        grown or cultured in the enrichment culture or cultures whose        distribution strongly correlates with a high methanogenesis        rate; and/or    -   (ii) identifying one or more microbes present in the sample or        samples whose distribution correlates with that of a methanogen        in the sample, or whose distribution correlates with that of a        methanogen(s) identified in step (c)(i).

In alternative embodiments the methods further comprise designing anutrient mix for optimizing growth of the methanogen(s) and/oroptimizing methanogenic activity,

wherein optionally the nutrient mix is at least initially based on oneor more requirements, or a range of requirements, of methanogenicmicrobes or microbes associated with methanogens as identified throughliterature searches, field observations of subsurface methanogenicenvironments and/or cultivation experiments,

and optionally the nutrient mix is also designed to decrease the amountof other (non-methanogenic) bacterial processes negatively affectingbiogas formation.

In alternative embodiments the methods further evaluating the effect ofnutrient concentration variations on methanogenesis rates in testcultures using endemic carbonaceous substrates. In alternativeembodiments the methods further comprise introducing the nutrient mix toa methanogenic microbial community, wherein optionally the methanogenicmicrobial community is in situ in a subsurface carbonaceous formation.

In alternative embodiments, the samples comprise a production water, orthe samples are taken from a core sample.

In alternative embodiments, the invention provides methods for improvingmethylotrophic biogas formation in situ in a subsurface carbonaceousformation comprising:

(a) administering one or more methanogenic organisms identified in amethod of the invention, or at least one synthetic consortium of theinvention, to the subsurface carbonaceous formation or formations, or

(b) administering one or more methanogenic organisms,

wherein optionally the methanogenic organisms comprise one or moremembers of the Archaea family, or are anaerobic organisms, or areautotrophs or chemoheterotrophs,

and optionally the methanogenic organisms comprise one or more membersof a genus selected from the group consisting of Methanolobus,Methanobacterium, Methanothermobacter, Methanogenium, Methanogenium;Methanofollis, Methanoculleus, Methanocorpusculum, Methanococcus,Methanocalculus, Methanobrevibacter and Methanosarcina,

and optionally the methanogenic organisms comprise: at least onesynthetic consortium of the invention, or one or more members selectedfrom the group consisting of:

-   -   Methanolobus bornbayensis    -   Methanolobus laylorii    -   Methanolobus profundi    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschalkii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofollis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeta concilii    -   Methanosaeta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospirillium hungatei    -   Methanothermobacter defluvii (Methanobacterium defluvii)    -   Methanothermobacter thermautotrophicus (Methanobacterium        thermoautoirophicum)    -   Methanothermobacter thermoflexus (Methanobacterium thermoflexum)    -   Methanothermobacter wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii

wherein optionally the She or more methanogenic organisms have beenenriched using the consensus and/or optimal nutrient mix identified in amethod of the invention,

wherein optionally the subsurface carbonaceous formation is modified tohave properties more like or similar to one or more properties of theoptimal nutrient mix

and optionally the subsurface carbonaceous formation or formationscomprises a coal formation, or a peat, or a lignite, or a bituminouscoal, or an anthracite coal, or a coal or a coal analogue(s) or aprecursor(s) thereof, heavy oil, asphaltenes, and/or an organic debris.

In alternative embodiments, the invention provides methods for improvingmethylotrophic biogas formation in situ in a subsurface carbonaceousformation or formations comprising:

(a) (1) administering one or more methanogenic organisms identified in amethod of the invention, or at least one synthetic consortium of theinvention, to the subsurface carbonaceous formation or formations,

wherein optionally the subsurface carbonaceous formation or formationscomprises a coal formation, or a peat, or a lignite, or a bituminouscoal, or an anthracite coal, a coal or a coal analogue(s) or aprecursor(s) thereof, heavy oil, asphaltenes, and/or an organic debris,or

(2) administering one or more methanogenic organisms,

wherein optionally the methanogenic organisms comprise one or moremembers of the Archaea family, or are anaerobic organisms, or areautotrophs or chemoheterotrophs,

and optionally the methanogenic organisms comprise one or more membersof a genus selected from the group consisting of Methanolobus,Methanobacterium, Methanothermobacter, Methanogenium, Methanogenium,Methanofollis, Methanoculleus, Methanocorpusculum, Methanococcus,Methanocalculus, Methanobrevibacter and Methanosarcina, or themethanogenic organisms comprise: at least one synthetic consortium ofthe invention, or one or more members selected from the group consistingof:

-   -   Methanolobus bornbayensis    -   Methanolobus taylorii    -   Methanolobus profundi    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschalkii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofollis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeta concilii    -   Methanosaeta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospirillium hungatei    -   Methanothermobacter defluvii (Methanobacterium defluvii)    -   Methanothermobacter thermautotrophicus (Methanobacterium        thermoautotrophicum)    -   Methanothermobacter thermoflexus (Methanobacterium thermoflexum)    -   Methanothermobacter wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii; or

(b) the method of (a), further comprising:

-   -   (i) applying (before, during and/or after administering the        organisms) to the subsurface carbonaceous formation an optimal        nutrient mix, or the optimal nutrient mix identified in any of        claims 3 to 15;    -   (ii) modifying (before, during and/or after administering the        organisms) the subsurface carbonaceous formation to have        properties more like or similar to one or more properties of the        optimal nutrient mix; or    -   (iii) a combination of both (i) and (ii).

In alternative embodiments of the methods, the methanogenic organismsand/or nutrient mix can (are designed to) decrease the amount of other(non-methanogenic) bacterial processes negatively affecting biogasformation, wherein optionally bacterial processes affectingsulfate-reduction and biohydrogen consumption via acetogenesis ornon-methanogenic hydrogenotrophic pathways are reduced.

In alternative embodiments, the invention provides, methods of enhancingmethanogenic rates in subsurface carbonaceous reservoirs comprisinginjecting one or more methanogenic organisms into the subsurfacecarbonaceous reservoir, wherein the one or more methanogenic organismscomprise: at least one synthetic consortium of the invention, one ormore members of the Archaea family, or are anaerobic organisms, or areautotrophs or chemoheterotrophs,

wherein optionally the subsurface carbonaceous reservoir comprises acoal formation, or a peat, or a lignite, or a bituminous coal, or ananthracite coal, a coal or a coal analogue(s) or a precursor(s) thereof,heavy oil, asphaltenes, and/or an organic debris.

In alternative embodiments of the methods, one or more methanogenicorganisms comprise one or more members of a genus selected from thegroup consisting of Methanolobus, Methanobacterium, Methanothermobacter,Methanogenium, Methanogenium, Methanofollis, Methanoculleus,Methanocorpusculum, Methanococcus, Methanocalculus, Methanobrevibacterand Methanosarcina, or the one or more methanogenic organisms comprise:at least one synthetic consortium of the invention, or one or moremembers selected from the group consisting of:

-   -   Methanolobus bornbayensis    -   Methanolobus laylorii    -   Methanolobus profundi    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschalkii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofollis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeta concilii    -   Methanosaeta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospirillium hungatei    -   Methanothermobacter defluvii (Methanobacterium defluvii)    -   Methanothermobacter thermautotrophicus (Methanobacterium        thermoautotrophicum)    -   Methanothermobacter thermoflexus (Methanobacterium thermoflexum)    -   Methanothermobacter wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii.

In alternative embodiments, the invention provides compositions,formulations, fluids, muds, or nutrient mixes for enhancing methanogenicrates in subsurface carbonaceous reservoirs comprising:

(i) one or more methanogenic organisms selected from the groupconsisting of a member of the Archaea family, an anaerobic organism, anautotroph, a chemoheterotroph or a combination thereof;

(ii) at least one synthetic consortium of the invention, or

(iii) the one or more methanogenic organisms of (i) and a consensusand/or optimal nutrient mix identified in a method of the invention,

wherein optionally the subsurface carbonaceous reservoir comprises acoal formation, or a peat, or a lignite, or a bituminous coal, or ananthracite coal, a coal or a coal analogue(s) or a precursor(s) thereof,heavy oil, asphaltenes, and/or an organic debris.

In alternative embodiments, the one or more methanogenic organismscomprise one or more members of a genus selected from the groupconsisting of Methanolobus, Methanobacterium, Methanothermobacter,Methanogenium, Methanogenium, Methanofollis, Methanoculleus,Methanocorpusculum, Methanococcus, Methanocalculus, Methanobrevibacterand Methanosarcina, or the one or more methanogenic organisms compriseone or more members selected from the group consisting of:

-   -   Methanolobus bornbayensis    -   Methanolobus taylorii    -   Methanolobus profundi    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschalkii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofollis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeta concilii    -   Methanosaeta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospirillium hungatei    -   Methanothermobacter defluvii (Methanobacterium defluvii)    -   Methanothermobacler thermautotrophicus (Methanobacterium        thermoautotrophicum)    -   Methanothermobacter thermoflexus (Methanobacterium thermoflexum)    -   Methanothermobacter wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii.

In alternative embodiments, the invention provides methods of creating amicrobial, composition to enhance methanogenic degradation ofcarbonaceous substrates comprising the following steps:

-   -   a. obtaining a sample from a subsurface carbonaceous        formation(s) of interest,

wherein optionally the sample comprises a water sample, or a productionwater sample;

-   -   and optionally the subsurface carbonaceous formation(s)        comprises a coal formation, or a peat, or a lignite, or a        bituminous coal, or an anthracite coal, or a coal or a coal        analogue(s) or a precursor(s) thereof, heavy oil, asphaltenes,        and/or an organic, debris,    -   b. using the sample to inoculate an enrichment culture        comprising a carbonaceous material of interest, and/or a        chemical analogue thereof, as carbon source;    -   c. incubating the enrichment culture until growth of an organism        is detected, wherein optionally the organism is a member of a        methanogenic community; and    -   d. introducing the cells detected in (c) into a subsurface        formation, wherein optionally the cells are introduced by a        method comprising injection at a well head.

In alternative embodiments, the enrichment culture is passaged intofresh medium at least one time. In alternative embodiments, the cellsare co-injected into the subsurface formation with an optimized nutrientmix.

In alternative embodiments, the invention provides products ofmanufacture, fluids, muds, bioreactors or surface or subsurfacereservoirs, for generating a biogas comprising: (a) production water,(b) a carbonaceous material of interest and/or a chemical analoguethereof as a carbon source; and (c) a composition Or a composition,formulation, fluid or nutrient mix of the invention, or at least onesynthetic consortium of the invention. In alternative embodiments, thecarbonaceous material or carbon source comprises or further comprises acoal, a bituminous coal, an anthracite coal, a volcanic ash, or alignite or a lignin or lignin-comprising composition, a coal or a coalanalogues or a precursors thereof, heavy oil, asphaltenes, and/or anorganic debris.

In alternative embodiments, the product of manufacture, fluid, mud,reservoir or bioreactor is contained in situ in a subsurface excavationor is contained in an artificial structure, or the product ofmanufacture or bioreactor is placed in or contained in a landfill or asubsurface carbonaceous reservoir or source. In alternative embodiments,the product of manufacture or bioreactor is a sand-pack bioreactor or acoal bioreactor.

In alternative embodiments, the biogas comprises methane, or the biogasmainly (or substantially) comprises methane.

In alternative embodiments, the following parameters are controlledand/or modified in the product of manufacture or bioreactor: i) type oforganic matter (plant vs algae derived), ii) thermal maturity of organicmatter (level of aromaticity and hence recalcitrance), iii) formationwater chemistry (i.e. salinity, pH, inorganic and organic waterchemistry), iv) temperature, and v) presence of appropriate syntrophicbacterial community able to provide specific methanogenic substrates.

In alternative embodiments, nutrients to enhance biogas formation areprovided to the product of manufacture or bioreactor. In alternativeembodiments, the nutrients to enhance biogas formation comprise metalsalts of compounds found in methylotrophic/bacterial enzymes,non-inhibitory level of alternate electron acceptors such as iron,manganese, or other nutrients and trace elements identified bycorrelating nutrient abundance to microbial growth/methane production.

In alternative embodiments, the environmental parameters in thebioreactor are modified to enhance biogas formation. In alternativeembodiments, the environmental parameters comprise formation orcomposition of water, pH of water (e.g., higher pH to the optimal rangeof the microbial association from culture experiments at the reservoirtemperature).

In alternative embodiments, the microbial populations and/or theenvironmental parameters in the bioreactor are manipulated or shiftedtowards more efficient coal/kerogen biodegrading, or more efficient CookInlet methanol/methyl-generating, or for increasing the methanogenesisrates.

In alternative embodiments, the products of manufacture, fluids, muds,reservoirs, bioreactors or surface or subsurface reservoirs comprise useof methylotrophic (methanol and other methyl-providing) substrates underneutral to slightly alkaline conditions to enhance biogas formation,wherein optionally the slightly alkaline conditions comprise conditionsof between about pH 7.5 to 9, or at least about pH 7.5, pH 8, pH 8.5, orpH 9.

In alternative embodiments, the products of manufacture, fluids, muds,reservoirs, bioreactors or surface or subsurface reservoirs comprise useof compositions and/or fluids to prevent or slow build up of volatilefatty acids such as propionic acid and/or to prevent or slow a pH dropthat would inhibit methanogenesis.

In alternative embodiments, a nutrient mixture or composition, or thecompositions and/or fluids, are introduced into a product ofmanufacture, fluid or bioreactor or a bioreactor reservoir throughinjection of a single bolus or through a continuous process. Inalternative embodiments, newly generated biogas is monitored and/ortraced from gas isotopes, using ¹⁴C-, ¹³C-, ²H- or ³H-enrichedmethanogenic substrates, and optionally the methanogenic substratescomprise bicarbonate, lignin and/or aromatic monomers.

In alternative embodiments, the invention provides methods for improvingmethylotrophic biogas formation in situ in a subsurface source orformation or an isolated, mined or excavated carbonaceous source orformation, comprising:

(a) administering to or contacting the subsurface source or formation orisolated, mined or excavated carbonaceous source or formation: at leastone synthetic consortium of the invention, or one or more methanogenicorganisms identified in a method of the invention, or

(b) administering to or contacting the subsurface source or formation orisolated, mined or excavated carbonaceous source or formation: one ormore methanogenic organisms,

wherein optionally the methanogenic organisms comprise one or moremembers of the Archaea family, or are anaerobic organisms, or areautotrophs or chemoheterotrophs,

and optionally the methanogenic organisms comprise one or more membersof a genus selected from the group consisting of Methanolobus,Methanobacterium, Methanothermobacler, Methanogenium, Methanogenium,Methanofollis, Methanoculleus, Methanocorpusculum, Methanococcus,Methanocalculus, Methanobrevibacter and Methanosarcina, or themethanogenic organisms comprise one or more members selected from thegroup consisting of:

-   -   Methanolobus bornbayensis    -   Methanolobus taylorii    -   Methanolobus profundi    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschalkii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofollis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeta concilii    -   Methanosaeta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospirillum hungatei    -   Methanothermobacter defluvii (Methanobacterium defluvii)    -   Methanothcrmobuctpr thermautotropliicm (Methanobacterium        thermoautotrophicum)    -   Methanothermpbacter thermoflexus (Methanobacterium thermoflexum)    -   Methanothermobacter wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii

wherein optionally the one or more methanogenic organisms have beenenriched using the consensus and/or optimal nutrient mix identified inany of claims 3 to 15, or the composition, formulation, fluid ornutrient mix of any of claims 22 to 24,

wherein optionally the subsurface carbonaceous formation is modified tohave properties more like or similar to one or more properties of theoptimal nutrient mix

and optionally the subsurface carbonaceous formation or formationscomprises a coal formation, or a peat, or a lignite, or a bituminouscoal, or an anthracite coal.

In alternative embodiments, the invention provides methods forprocessing a heavy oil, or decreasing the viscosity of a heavy oil byconverting high molecular weight hydrocarbons into lower molecularweight hydrocarbons, or converting a heavy oil, a bitumen, a tar-sand,or equivalents, to a less viscous from, or to a gaseous light gas, gasand/or diesel product, wherein optionally the less viscous form of theheavy oil, bitumen, tar-sand or equivalents comprises substantially fromC1 to about C24 hydrocarbons, comprising:

(a) injecting: at least one synthetic consortium of the invention,and/or one or more methanogenic organisms, into a subsurfacecarbonaceous reservoir comprising a heavy oil, a bitumen, a tars-and, orequivalents, or

(b) contacting the heavy oil, a coal, a bitumen, a tars-and, orequivalent with: at least one synthetic consortium of the invention, ora composition comprising one or more methanogenic organisms, whereinoptionally the contacting is in situ (e.g., in a ground formation or asubsurface carbonaceous reservoir), or a man-made reservoir or productof manufacture, or an excavated, mined, drilled or isolated heavy oil,bitumen, tar-sand, or equivalent,

wherein the one or more methanogenic organisms comprise one or moremembers of the Archaea family, or are anaerobic organisms, or areautotrophs or chemoheterotrophs,

wherein optionally the subsurface carbonaceous reservoir comprises acoal formation, or a peat, or a lignite, or a bituminous coal, or ananthracite coal.

In alternative embodiments of the methods, the one or more methanogenicorganisms comprise one or more members of a genus selected from thegroup consisting of Methanolobus, Methanobacterium, Methanothermobacter,Methanogenium, Methanogenium, Methanofollis, Methanoculleus,Methanocorpusculum, Methanococcus, Methanocalculus, Methanobrevibacterand Methanosarcina, or the one or more methanogenic organisms compriseone or more members selected from the group consisting of:

-   -   Methanolobus bornbayensis    -   Methanolobus taylorii    -   Methanolobus profundi    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschaikii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofolis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeia concilii    -   Methanosaetta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospiriilium hungatei    -   Methanothermobacler defluvii (Methanobacterium defluvii)    -   Methanothermobacter thermauiotrophicus (Methanobacterium        thermoautoirophicum)    -   Methanothermobacler thermoflexus (Methanobacterium        thermoflexuin)    -   Methanothermobacler wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii.

In alternative embodiments, the invention provides methods comprising anintegrated process for optimizing biogas generation from subsurfaceorganic matter-rich formations (coal and/or other organic-containingrocks), comprising one or more, or all, the following steps:

(a) a microbial collection procedure conducive to acquiring both deepmicrobial community surveys (DNA/RNA analyses) and cultured isolates ofkey living microorganisms;

(b) identification of specific target microbial associations capable ofrapidly transforming organic matter to biogas, using empiricalcorrelation of the microbial profiling data (e.g., from454-pyrosequencing) to key geochemical parameters using an integratedmulti-disciplinary data-set;

(c) simultaneous, identification of unfavorable endemic microbes orconditions showing negative correlation to, biogas formation, asidentified in 6b above;

(d) use of microbial evaluation tools, to further identify the specificactive microbes critical to biogas growth (or inhibition) out of theempirically identified microbial targets;

(e) rock characterization of both indigenous organic carbon-richsubstrates and inorganic mineralogy affecting the water-injectate recipecomposition for enhanced biogas formation and selection of substraterocks;

(f) further optimization of the proposed injectate-water chemistry froma matrix of laboratory enrichment experiments to promote subsurfacebiogas production without activating deleterious microbial effects atthe reservoir temperature of the target field) and subsequentflow-through core experiments using the water-injectate recipe ontargeted rock cores;

(g) geochemical modeling of the solution stability to account forundesired precipitation of minerals due to interactions between in-situformation water, the injectate and in-situ mineral phases;

(h) modeling fluid transport within, the reservoir structure anddelivery mechanisms to successfully spread the water-soluble amendmentsand cultured microbes to the target formations;

(i) modeling of transport of the newly generated microbial methanewithin the reservoir towards the gas column and the producing wells;

(j) field implementation of the biogas production process; and/or

(k) field monitoring of biogas production and collateral microbial/waterchanges,

In alternative embodiments, the invention provides methods oftransforming a carbonaceous substrate, a carbonaceous material or acarbon source into a lower molecular weight (MW) compound using asynthetic microbial consortia comprising the steps of:

-   -   a. Providing a plurality of samples that comprise a carbonaceous        substrate and microbial communities;    -   b. Determining the composition of the microbial community in        each sample;    -   c. Identifying a consortium (a grouping) of microbes whose        abundance correlates with transformation of the carbonaceous        substrate;    -   d. Assembling a synthetic consortium by combining individual        pure cultures in a strain collection;    -   e. Combining the synthetic consortium with a carbonaceous        substrate to convert it to a higher value and lower molecular        weight product;

and optionally the samples are enrichment cultures incubated with thecarbonaceous substrate.

In alternative embodiments of the method, the carbonaceous substrate,carbonaceous material or carbon source comprises or further comprises acoal, a bituminous coal, an anthracite coal, a volcanic ash, or alignite or a lignin or lignin-comprising composition, a coal or a coalanalogues or a precursors thereof, heavy oil, asphaltenes, and/or anorganic debris.

In alternative embodiments, the invention provides methods forincreasing or stimulating a coal to methane conversion rate, comprising:

(a) injecting: at least one synthetic consortium of the invention,and/or one or more methanogenic organisms, into an isolated (e.g., outof ground, mined or excavated) or a subsurface carbonaceous or coalreservoir or a source comprising a coal, a bitumen, a tar-sand or anequivalent, or

(b) contacting the isolated or subsurface carbonaceous or coalreservoir, or coal, bitumen, a tar-sand, or equivalent, with: at leastone synthetic consortium of the invention, or a composition comprisingone or more methanogenic organisms, wherein optionally the contacting isin situ (e.g., in a ground formation or a subsurface carbonaceousreservoir), or a man-made reservoir or product of manufacture,

wherein the one or more methanogenic organisms comprise one or moremembers of the Archaea family, or are anaerobic organisms, or areautotrophs or chemoheterotrophs,

wherein optionally the subsurface carbonaceous reservoir comprises acoal formation, or a peat, or a lignite, or a bituminous coal, or ananthracite coal.

In alternative embodiments, wherein the one or more methanogenicorganisms comprise one or more members of a genus selected from thegroup consisting of Methanolobus, Methanobacterium, Methanothermobacter,Methanogenium, Methanogenium, Methanofollis, Methanoculleus,Methanocorpusculum, Methanococcus, Methanocalculus, Methanobrevibacterand Methanosarcina, or the one or more methanogenic organisms compriseone or more members selected from the group consisting of:

-   -   Methanolobus bornbayensis    -   Methanolobus taylorii    -   Methanolobus profundi,    -   Methanolobus zinderi    -   Methanobacterium bryantii    -   Methanobacterium formicum    -   Methanobrevibacter arboriphilicus    -   Methanobrevibacter gottschalkii    -   Methanobrevibacter ruminantium    -   Methanobrevibacter smithii    -   Methanocalculus chunghsingensis    -   Methanococcoides burtonii    -   Methanococcus aeolicus    -   Methanococcus deltae    -   Methanococcus jannaschii    -   Methanococcus maripaludis    -   Methanococcus vannielii    -   Methanocorpusculum labreanum    -   Methanoculleus bourgensis (Methanogenium olentangyi &        Methanogenium bourgense)    -   Methanoculleus marisnigri    -   Methanofollis liminatans    -   Methanogenium cariaci    -   Methanogenium frigidum.    -   Methanogenium organophilum    -   Methanogenium wolfei    -   Methanomicrobium mobile    -   Methanopyrus kandleri    -   Methanoregula boonei    -   Methanosaeta concilii    -   Methanosaeta thermophila    -   Methanosarcina acetivorans    -   Methanosarcina barkeri    -   Methanosarcina mazei    -   Methanosphaera stadtmanae    -   Methanospirillium hungatei    -   Methanothermobacter defluvii (Methanobacterium defluvii)    -   Methanothermobacter thermautotrophicus (Methanobacterium        thermoautoirophicum)    -   Methanothermobacter thermoflexus (Methanobacterium thermoflexum)    -   Methanothermobacter wolfei (Methanobacterium wolfei), and    -   Methanothrix sochngenii.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Otherfeatures, objects and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims. Reference will now be made in detail tovarious exemplary embodiments of the invention, examples of which areillustrated in the accompanying drawings. The following detaileddescription is provided to give the reader a better understanding ofcertain details of aspects and embodiments of the invention, and shouldnot be interpreted as a limitation on the scope of the invention. A morecomplete understanding of the present invention and benefits thereof maybe acquired by referring to the follow description taken in conjunctionwith the accompanying drawings in which:

FIG. 1 graphically illustrates the Rank abundance plot of 16S rRNA genesequences isolated from production water of gas wells from the CookInlet, Alaska. DNA sequences belonging to the genus Methanolobus areshown as highlighted bars. The relative proportions of methanogens thatutilize one or more of the three methanogenic pathways are indicated(inset).

FIG. 2: graphically illustrates the Distribution of Archaeal populationsalong pH gradient (bottom panel) in the Cook Inlet wells. Note high pHand high methane production rates typically coincide with significantfraction of Methanolobus. High positive correlation betweenfield-measured pH of formation water and Methanolobus population (datalog transformed and Z-scored) is indicated in the inset.

FIG. 3: graphically illustrates Methanogenesis fates fromcoal/lignin/lignin monomers and fractions of methanogens split intosubstrate-specific categories. Microbial populations from two wells withlargest fraction of obligate methyl/methanol utilizers (40-3 and 21-5)obtained highest rates of methanogenesis expressed as mL of CH₄ per L ofmedium per day. Wells 21-1, 21-4, and 40-1 from FIG. 2 not shown due tolittle or no methane production.

FIG. 4: is a Schematic of an exemplary process of this invention forcreating optimized chemical recipe for enhanced microbialmethanogenesis.

FIG. 5: is a Schematic of an exemplary process of this invention forcreating optimized nutrient and microbial mixes or compositions, e.g.,nutrient and microbial mixes or compositions of the invention.

FIG. 6: graphically illustrates a Visual representation of formationwater adjustment to the optimized recipe. Example formation watercomposition co-produced from one of the Cook Inlet gas wells. Requiredadjustment of parameters is represented by arrows.

FIG. 7: graphically illustrates an Example of optimization methaneproduction from Cook Inlet rock material by varying single parameter insand-pack incubations (total methane produced after 6 weeks ofincubation).

FIG. 8: graphically illustrates a Comparison of methane production ratefrom Cook Inlet rock material without and with optimized nutrientaddition. Production water from 40-3 well. Production of methane withoutnutrient addition is significantly slower after 42 days of incubation.Each data point represents a triplicate set of sand pack tubes.

FIG. 9: graphically illustrates a Methane production from the Cook Inletrock material; sand-pack incubations using cell additions of the CookInlet consortia grown on mixture of the Cook Inlet coal, lignin andlignin monomers: FIG. 9( a) addition of microbial consortium from thesame well and grown on coal/lignin/lignin monomers mixture (well 40-3had the highest rate, sec FIG. 3, hence this consortium was used asinoculum) enhances the methanogenesis rate over tubes with optimizedchemical recipe only, but no cell additions; FIG. 9( b) addition ofcoal/lignin/lignin monomers mixture-grown consortium to formation waterfrom well that originally had very low methane production (FIG. 3). Notethat adding coal/lignin/lignin monomers-grown consortia to productionwater from different basin (California) also successfully increasesmethane production from the same rock material. Each data pointrepresents a triplicate set of sand pack tubes.

FIG. 10: graphically illustrates Introduction of a pure Methanolobustaylorii culture increases methane production in model sand packincubations. Each data point represents a triplicate set of sand packtubes.

FIG. 11: schematically illustrates an exemplary mechanism or method ofthe invention for enhancement of biogas generation in a highly permeableformation with predominantly dispersed organic debris and thin beddedcoals; in one embodiment, injection of nutrient-amended injectate intothe water leg down dip and/or into production-induced water legstimulates biogas generation and migration up-dip towards gas cap andproduction well.

FIG. 12: is a Schematic representation of an exemplary method of theinvention comprising nutrient and microbe injection in a fieldapplication. FIG. 12(A) Representation of well injection systemincluding existing injection water systems, concentrated nutrientstorage tank, nutrient mixing tank, and injection line for injectingdilute nutrient mixture. FIG. 12(B) One example of a Batch Mixing tank Aand Storage tank B for storage and mixing of concentrated nutrientsolutions up to 250 bbls per batch.

FIG. 13: graphically illustrates Field measurements of redox potential(black bats) and oxygen saturation (gray bars) of produced water fromvarious producing wells and from the vacuum truck which collects waterfrom all wells for disposal into injection well.

FIG. 14: graphically illustrates Test results of effectiveness andconcentration of sodium hypochlorite (NaOCl) required to control biomassformation in injection line and injection well-bore: FIG. 14A, Level ofbiomass before treatment with 0.6% NaOCl solution at various oxygenlevels and nutrient conditions; FIG. 14B, Level of biomass immediatelyafter treatment with 0.6% NaOCl solution at various oxygen levels andnutrient conditions. CFU/mL=Colony-forming units per milliliter.

FIG. 15: graphically illustrates Biomass development in an exemplary(1×) concentration nutrient recipe of the invention, and in an exemplary“excess (25×) concentration nutrient recipe” of the invention: FIG. 15A.Biomass level over time in cultures incubated at 10° C. and 20° C. in25× excess concentration nutrient recipe; FIG. 15B, Biomass level incultures incubated at 25° C. in standard (1×) concentration of recipe.CFU/mL=Colony-forming units per milliliter.

FIG. 16: A schematic illustration representation of a three-dimensionalgeocellular model showing the biodegradable coal fraction in one layer.Lighter color indicates a higher percentage, of biodegradable coal.Arrow in picture points North. This model was used to simulate, thevolume and flow of biogenic gas generated from the addition of optimizednutrients and microbial additions.

FIG. 17: graphically illustrates the results from simulation, ofmultiple biogas generation rates/volumes using compositions and methodsof the invention, showing tracer concentration observed at themonitoring well over time, starting at 5 months after start of gasinjection. Travel time between injection well and monitor well isreduced with higher biogas generation rates/volumes.

FIG. 18: graphically illustrates Level of trace compounds in monosodiumphosphate from two different commercial vendors.

FIG. 19: graphically illustrates Effect of NaOCl solution in absence (A)and presence (B) of oxygen on viability of microbial population.CFU/mL=Colony-forming units per milliliter.

FIG. 20: graphically illustrates Development of biomass in (A)concentrated nutrient solution (25×) and (B) standard concentration ofnutrient (1×). CFU/mL=Colony-forming units per milliliter.

FIG. 21: graphically illustrates graphically illustrates Effect ofoxygen-scavenging compounds on redox potential of produced waterpreviously exposed to oxygen. mV=millivolts.

FIG. 22: is a Schema describing an exemplary method of the invention (ora method used to make a composition of the invention) comprising stepsof finding, assembling and deploying a synthetic consortium of microbes.

FIG. 23: illustrates a Two-dimensional cluster analysis of 16S rRNAgenes from biogenic gas samples. The numerical values in each cell ofthe array represent the number of times a specific 16S rRNA genesequence was identified in that sample. The values in the first columnare the sum of the occurrences of each sequence in all samples (Note,this view is truncated and does not include all of the samples or all ofthe sequences identified). Each column in the array represents a singlesample. Each row in the array is a unique 16S rRNA gene sequence whichserves as a proxy for a unique microbe. The columns are rearranged(clustered) according to the microbial community present in that samplesuch that samples with similar microbial communities are groupedtogether. The rows are clustered according to the abundance distributionof each sequence across the samples. Thus, sequences with similardistributions are grouped.

FIG. 24: graphically; illustrates Methane production in sandpacksincubations, supplemented with additional cells including the exemplary“Consort-ABS1” composition of the invention.

DETAILED DESCRIPTION

Turning now to the detailed description of the arrangement orarrangements of the one or more embodiments of the invention, it shouldbe understood that the inventive features and concepts may be manifestedin other arrangements and that the scope of the invention is not limitedto the embodiments described or illustrated. The scope of the inventionis intended only to be limited by the scope of the claims that follow.

The invention provides compositions and methods for commercial biogas,e.g., methane, production. In alternative embodiments, the inventionprovides compositions and methods for methanol-utilizing methanogenesis,or “methylotrophic”-conversion, including utilizing methylamines andother methyl-containing intermediates.

The inventors have successfully demonstrated faster, commercial biogas(e.g., methane) production rates under highly favorable laboratoryconditions by enhancing the microbial environment, e.g., by varying pH,microbe and nutrient supplementation of water. The inventors havedemonstrated that biogenic gas fields in the Cook Inlet (Alaska) have asurprisingly significant contribution from a third, equally importantand often disregarded pathway—methanol-utilizing methanogenesis, or“methylotrophic”-conversion, which also can include substrates such asmethylamines and other methyl-containing intermediates. In alternativeembodiments the invention provides compositions and methods comprisinguse of methanol-utilizing methanogenesis, which also can include, use ofsubstrates such as methyl amines and other methyl-containingintermediates.

In alternative embodiments the invention provides an integrated processfor optimization of biogas (e.g., methane) generation in subsurfaceorganic matter-rich formations (e.g., man made formations, such aslandfills, or natural formations such as coal formations, shale,sandstone or limestone with organic debris or oil) via themethylotrophic pathway.

In alternative embodiments the ultimate goal of this biogas applicationis to extend the productive field-life of sub-surface biogenic-gasassets. In alternative embodiments, field implementation of biogasproduction is based on an integrated microbial-substratecharacterization, including all or some of the following steps: (1) amicrobial collection procedure conducive to both deep microbialcommunity surveys (DNA/RNA analyses), culturing and isolation of livingmicroorganisms; (2) identification of specific target microbialassociations capable of rapid transformation of subsurface organicmatter to biogas via e.g. the methylotrophic pathway, using empiricalcorrelation of microbial profiling (in alternative embodiments usingpyrosequencing) data to key geochemical parameters and targetedincubations (e.g. with lignin or other coal-analogues or precursors);(3) simultaneous identification of unfavorable endemic microbes orconditions showing negative correlation to biogas formation using thesame information as in step #2; (4) formation characterization of bothindigenous organic carbon-rich substrates and inorganic mineralogyaffecting the water-injectate composition for biogas formation(including core-water-microbe experiments); (5) optimization of aninjectate water chemistry (especially water pH and essential nutrients)and microbiology (selected isolates or pre-grown success fillcommunities obtaining high methanogenesis rates with targeted coal andcoal analogues) to promote subsurface biogas production at the reservoirtemperature of the target field); (6) investigation and modeling ofdelivery mechanisms to successfully spread the water-soluble amendmentsand cultured microbes to the target formations; and (7) fieldimplementation of any one or all of these steps in e.g., a biogasproduction process.

In alternative embodiments, the compositions and methods of theinvention identify, mimic and/or manipulate the combination ofparameters that result in the specificity of a methanogenic pathway inthe subsurface, including e.g., any one or a combination of parameters,for example: i) type of organic matter (e.g., plant versus (vs) algaederived), ii) thermal maturity of organic matter (e.g., level ofaromaticity and hence recalcitrance), iii) formation water chemistry(e.g., salinity, pH, inorganic and organic water chemistry), iv)temperature, and v) presence of appropriate syntrophic bacterialcommunity able to provide specific methanogenic substrates.

In alternative embodiments, the invention provides compositions, e.g.,nutrient mixes, and methods of enhancing biogenic methane productionthrough the creation of customized nutrient amendments (e.g.,supplements, mixes and the like), wherein the compositions and methodscan be used to specifically stimulate (or inhibit, as appropriate)functionally important constituents of a microbial community responsiblefor biogas formation (or responsible for inhibition of optimal biogasproduction). In alternative embodiments, the invention providesmicrobial compositions (including bioreactors, which include subsurfacereservoirs) to augment microorganisms involved in the methanogenicdegradation of recalcitrant organic matter or to introduce new microbialfunctionalities into a reservoir to initiate or stimulate this process.

In alternative embodiments, the invention can identify a microbialcommunity present in a subsurface carbonaceous reservoir, e.g., bynucleic acid (e.g., DNA, RNA) characterization, e.g., by nucleic acidsequencing, hybridization, PCR and the like, to determine orcharacterize the microbes present (optionally including their relativeabundance); and in alternative embodiments a customized nutrient mixtureof the invention comprises, or is based on: (1) published nutrientrequirement values that are weighted toward the more abundant andimportant (relative to the targeted methanogenic pathway) organisms; and(2), field observations about specific reservoir conditions (e.g. waterchemistry, well production, etc.). In alternative embodiments, theresulting customized nutrient composition of the invention is introducedto a reservoir through an injection process at the well head, or is usedin a bioreactor of the invention. In alternative embodiments, abioreactor of the invention includes any subsurface space or reservoir,such as a man-made subsurface reservoir.

Organisms that participate in a given biogeochemical process or pathwaymake up consortia and might be expected to be coordinately distributedin the environment. In other words, the members of a given consortiumwill tend to be found together. The degree to which these microbes arefound together is expected to be a function of the obligate nature oftheir metabolic relationship. For example, two syntrophic organisms thatonly utilize a single carbon substrate and that were absolutelydependent upon each other to metabolize the substrate would display thestrongest coordinated distribution since neither partner could exist orproliferate without the other (e.g., sulfate reducing bacteria withanaerobic methane oxidizers or potentially a methanol producingbacterium with obligate methylotrophic methanogen such as Methanolobus).In other cases where two syntrophic organisms had similar dependenciesupon one another for a given substrate, but had additional substratesthat they could utilize independently of the syntrophic partner, woulddisplay a much less tightly linked environmental distribution. Theorganisms of this latter example are expected to have a coordinateddistribution among environments where syntrophy was necessary formetabolism of the prevailing substrates. An example of this situation isin subsurface accumulations of coal.

This tendency of members of a given consortium to be found together isan attribute that can be used to identify microbes that work together ina given biodegradative process such as the conversion of coal intomethane (Ashby 2003). By identifying the key microbial players thatperform a process of interest in a particular environment, they can bespecifically re-introduced into an environment to enhance the rate andspecificity of said process.

In alternative embodiments, a consortium is a group of two or more (aplurality of) microorganisms that participate in a common ecological orbiosynthetic or biodegradative process, e.g., a biogeochemical process.During biogenic gas formation, microbes can participate in the samebiogeochemical process or metabolic pathway. Oftentimes, these microbesare able to perform distinct steps of the same metabolic, biochemical orbiodegradative pathway. In some embodiments, the term “consortium”defines a group of (a plurality of) microorganisms that participate inthe same biogeochemical cycle, such as the conversion of a coal to amethane, or biodegradation of a heavy oil; and in alternative embodimentconsortiums of the invention are used to convert a coal, or a coalanalogue(s) or a precursor(s) and the like to a methane, or biodegradean oil or a heavy oil and the like. In other embodiments, the term“consortium” is defined as a group of microorganisms that participate ina unified set of biochemical reactions, such as in biogeochemicalcycles.

In alternative embodiments, species describes a taxonomic rank of anorganism. In alternative embodiments species are classified based ontraits such as similarity of DNA, morphology or ecological, niche. Inalternative embodiments species are grouped using statistical analysisof DNA sequences or markers to determine the relatedness of two or morebacterial or Archaeal microorganisms. In one embodiment, two or moreorganisms are classified as members of the same species when analignment; of the 16S rRNA gene sequences reveals about 5% or lessdifference (95% identity) at the nucleotide level, about 4% or lessdifference (96% identity) at the nucleotide level, about 3% or lessdifference (97% identity) at the nucleotide level, about 2% or lessdifference (98% identity) at the nucleotide level, or about 1% or lessdifference (99% identity) at the nucleotide level.

In alternative embodiments, synthetic consortium are a set, of microbeswhere each one exists in pure culture and are combined to form a definedmixture or consortium of microbes that can perform a particular, usefulfunction. In one embodiment, a synthetic consortium comprises two ormore cultured species available from commercial and/or unique isolatedcultures where the cultured species are selected to performcomplementary processes in a geochemical or biogenic gas pathway, in oneembodiment, a synthetic consortium of microbes comprises two or moreuncultured species that are combined by physical means where thecultured species are selected to perform complementary processes in ageochemical or biogenic gas pathway.

In alternative embodiments, syntrophs are organisms that utilizeproducts from another organism. In one embodiment, two or more microbesmay be dependent upon each other to perform a biochemical reaction,generate an essential product, or produce a substrate or cofactor.

In alternative embodiments, biochemical and geochemical compositionsundergo one or more chemical transformations. In one embodiment, asubstrate is transformed when it undergoes a biochemical reactionthrough the action of enzymes produced by biological organisms, forexample, by practicing a method of this invention. In anotherembodiment, the transformation involves one or more catabolic reactionswhere the result of the process or pathway is reduction in the molecularweight of the substrate.

In alternative embodiments, upgrading heavy oil as used herein describesthe process of lowering the boiling point of a composition that mayinclude heavy crude oil, bitumen, tars, and other high viscosityhydrocarbons. The viscosity of crude oil or tar usually by reducing themolecular weight of its constituents, increasing aromatic components,removing volatile fatty acids, increasing the gas to oil (GOR) ratio,addition of solvents, increasing the hydrogen content, and otherprocesses where viscosity is decreased. In one embodiment the viscosityof the heavy oil is decreased by convening high molecular weighthydrocarbons into lower molecular weight hydrocarbons. In anotherembodiment, heavy oils, bitumens, tarsands and the like are converted toless viscous or gaseous light gas, gas and diesel range products fromC1-C24 hydrocarbons.

Identifying Relevant Consortium and its Members

In one embodiment, the composition of microbial communities isdetermined or profiled from samples that have been in contact with coalor other carbonaceous material of interest. These samples will includeenvironmental samples such as production water, formation water, coresamples, drill cuttings, water, sediment or soil. Optimally, the sampleswould contain the same carbonaceous material that was the subject ofinvestigation to find microbes capable of transforming into a highervalue product. For example, samples would be chosen that contained coalthat had a similar level of maturity as that in the target basin.

In another embodiment, the microbial communities present in thesesamples are used to inoculate cultures comprising a carbon source,essential nutrients (including vitamins, trace metals and a source ofphosphorus; sulfur and nitrogen), and optionally including a buffer tomaintain pH, a reducing agent (sodium sulfide, sodium dithionite,dithiothreitol, thioglycollate or cysteine), a redox indicator (e.g.resazurin or methylene blue) and a terminal electron acceptor (e.g.oxygen, nitrate, sulfate, Fc(III), Mn(IV), carbon dioxide, oranthraquinone disulfonate (AQDS)). Anaerobic culture conditions,enrichment methods and medium formulations are widely known to thoseskilled in the art and may be practiced in a variety of ways such asthose described by Shelton and Tiedje (Shelton and Tiedje 1984). Thecarbon source for the enrichments would be of the same type such as coalor asphaltenes as described above.

In an alternative embodiment, the enrichment cultures are maintained inscrum vials. At various time points in their incubation, the enrichmentcultures would be tested for growth and metabolism. Cell growth isassayed by microscopic cell counts or by measuring optical density at550 or 600 nm wavelength in a spectrophotometer. Metabolism is measuredby gas production where the volume of gas produced is determined with apressure transducer (Shelton and Tiedje 1984) and the type of gas (e.g.CH4, H2, or CO2) is determined by gas chromatography. The transfer ofelectrons to AQDS and the resulting color change from clear to orange,can also be used as a measure of metabolic activity. Additionally,consumption of the carbonaceous substrate can indicate metabolicactivity.

In yet another embodiment, DNA is extracted from the enrichment culturesto characterize the microbial community at the beginning of incubationand after growth and/or metabolism is detected. This community analysiscan be done repeatedly to characterize community changes during theperiod of incubation and can be tracked together with the geochemicalchanges of the medium and gaseous headspace. After the enrichmentcultures exhaust nutrients as evidenced by a reduction in growth rate ormetabolic activity, the cultures are optionally passaged into freshmedium using a dilution factor such as 1 ml of original culture dilutedinto 100 mls of fresh medium. The methods described above to determinegrowth and metabolism are repeated for subsequent passages. Thisexercise of repeated growth and transfer to fresh medium can also beperformed in bioreactors, fermenters or chemostats and will have theeffect of diluting away (‘washing out’) members of the community thatare not involved in metabolizing the target substrate. At the same limeconsortium members that are involved in metabolizing the substrate willbecome established if they are able to increase their cell numbers tooffset their dilution during culture passaging or through the outflow ofmedium in a chemostat.

An exemplary method of the invention for determining the microbialcommunity composition can comprise any of the methods known to thoseskilled in the art; such as, e.g., DNA sequencing of all or a portion of16S rRNA genes, by hybridization of sample derived DNA to immobilizedoligonucleotides or PCR generated probes (i.e. DNA microarrays),quanlitative PCR (qPCR) analysis, separation of DNA fragments such asterminal restriction fragment length polymoqhism (T-RFLP) analysis or bynon-DNA-based methods such as fatty acid methyl ester (FAME) analysis.For DNA-based profiling methods, genomic DNA is isolated by any of anumber of methods or commercially available kits that would result inthe efficient recovery of DNA with a minimal level of introduced biasFor DNA sequence profiling of 1.6S rRNA genes, ‘universal’ primers canbe utilized to PCR amplify a portion of the gene that includes variableregions. Limiting the number of PCR cycles can reduce biases andartifacts that might occur.

In one embodiment, the microbial community composition profile datadetermined through the use of culture independent, molecular surveysdescribed, above, optionally in the form of number of copies of eachdistinct 16S rRNA gene sequence detected from each sample, is thenanalyzed to detect the distribution patterns of microbes amongst thesamples tested. As indicated above, microbes that participate in thesame biodegradative or metabolic pathway and thus, members of a commonmicrobial consortium will tend to be found together in the environment(including samples derived therefrom). This relationship can also bededuced from abundance data in culture independent surveys (Ashby 2003).

In one embodiment, to identify potential relationships that existbetween environmental microbes as indicated by their tendency to becoordinately distributed in the environment, the data is first logtransformed. Log transformation tends to make microbial distributiondata more normally distributed which may result from the logarithmicnature of microbial growth. Log transformed microbial distribution datacan then be compared between different 16S rRNA gene sequence detectedusing correlation analysis (e.g. Pearson). Operationally, a distancematrix is constructed where, the distribution of every sequence iscorrelated with that of every other sequence. The results can then begraphically represented using hierarchical clustering algorithms such asWard's method. Computer software programs are widely available toperform this analysis such as PC-ORD (Gleneden Beach, Oreg.). Thisexercise will reveal groups of sequences that tend to be found together(see example below). Comparison of the distribution of the group as awhole to the transformation or metabolic activity observed in thesamples (or enrichment cultures) will provide further evidence as to themetabolic functional capability of the consortium.

In alternative embodiments, the members of a consortium are identifiedfrom microbial community surveys using distance metrics that includeEuclidean distance, Chi square, city block, and ordination methods thatinclude PCA, Bray-Curtis, and nonmetric multidimensional scaling (NMS orNMDS).

Utilizing Consortium to Enhance Transformation Rate of a CarbonaceousSubstrate

In alternative embodiments, consortium of microbes to be utilized toenhance methanogenesis rates can be prepared by multiple strategies. Oneapproach involves systematically isolating in pure culture all of themembers of the consortium of interest. The individual consortium membersare then combined into a synthetic consortium which can then be testedfor metabolism of the substrate of interest and/or utilized for thecommercial scale conversion of a carbonaceous substrate into a highervalue-lower molecular weight product.

In alternative embodiments, methods and medium formulations forisolating environmental microbes in pure form comprise those known inthe art. In alternative embodiments, for consortia that would ultimatelybe deployed in the subsurface where oxygen is absent, anaerobiccultivation methods are used. Samples or enrichment cultures thatpossess the microbes of interest are diluted and plated onto a varietyof solid medium containing different nutrient combinations to obtainsingle colonies. At least one of the medium formulations should containthe carbonaceous substrate of interest. Parameters such as saltconcentration and pH should be as consistent as possible with theoriginal sample where the organisms of interest, were present oradjusted to optimize growth of targeted microbes and enhancement oftargeted metabolic process. Oftentimes environmental microbes aredifficult, if not impossible, to cultivate and their isolation requiresthe use of alternative strategies such as dilute nutrients and differentmedium solidifying agents (e.g., sec Connon and Giovannoni 2002; Sait,Hugenholtz et al. 2002).

In alternative embodiments, microbial colonies that appear on platesfollowing incubation should be picked and re-streaked onto fresh mediumat a low enough density to obtain new, well resolved colonies. Thiscolony purification procedure can be repeated to reduce the risk ofcolonies being comprised of multiple species. The resulting coloniesshould display a uniform morphology consistent with a homogenouspopulation of organisms. In alternative embodiments, a colony is pickedand grown up either in liquid culture or as a patch on the same mediumtype. The resulting culture is then frozen at −80° C. and/or freezedried for archival purposes. DNA from the cells from the same culture isextracted for identification by sequencing its 16S rRNA gene.

In alternative embodiments, a second approach is to utilize enrichmentcultures as described above to select for a consortium with theproperties of interest while at the same time selecting against microbesthat do not participate in the process. This approach is utilized whensome members of the consortium of interest cannot be cultivated in pureform. Organisms that are expected to fall into this category includeobligate syntrophs which by definition cannot be grown in pure culturein the absence of their syntrophic partner. While this approach is notas preferable as the pure culture route that can produce a community ofexactly the members desired, it can lead to a highly enriched culturefor the organisms with the metabolic potential of interest. Suchsuccessful culture might be further tested to identify tightly boundsyntrophic associations. Subsequent cultivation may allow isolation ofthese tight associations, their phylogenetic confirmation by DNAextraction, and their storage for further lab and commercial use.

In alternative embodiments, additional methods of assembling a syntheticconsortium of the invention involve physically separating cells presentin a sample using methods such as fluorescence activated cell sorting(FACS). The cells of interest can be specifically labeled withfluorescent labeled probes and fluorescent in situ hybridization (FISH)without using fixatives. Other methods to physically separate cells ofinterest include optical tweezers or through the use of antibodies thatspecifically recognize determinants on the cell of interests surface.

In alternative embodiments, synthetic consortium of the inventioncomprise a mixture of cells each derived from pure isolates or a highlyenriched consortium, which optionally can be derived from a selectivegrowth, and then optionally can then be introduced into a subsurfacereservoir or other environment containing the carbonaceous substrate ofinterest, where optionally the consortium has been selected for growthand metabolic performance under the specific environmental conditionswith the goal to convert the substrate to a higher value product.

One embodiment provides methods for increasing commercial biogasproduction in a sub-surface environment. In another embodiment theinvention provides an integrated process for optimization of biogasgeneration including methane in subsurface organic matter-richformations including man made formations, such as landfills, surface orsubsurface bioreactors (in alternative embodiments, a bioreactor of theinvention or a bioreactor used to practice the invention includes anysubsurface space or reservoir, such as a man-made subsurface reservoir)and the like, or natural formations such as shale, coal, oil sands,bitumen, tar, oil, sandstone and limestone with organic debris or otherhydrocarbon rich formations via the methylotrophic pathway. Methods foranalysis and understanding of subsurface; microbial communitiesresponsible for conversion of coal and coal-like substrates intomethane, and for controlling geochemical conditions are provided. Thus,in alternative embodiments, compositions and methods to stimulatesubsurface methanogenesis pathways and to enhance the rates of biogasformation are provided.

In alternative embodiments, methods of the invention for increasingbiogas production extend the productive field-life of sub-surfacebiogenic-gas assets. In alternative embodiments, field implementation ofbiogas production is based on an integrated microbial-substratecharacterization, including all or some of the following steps: (1) amicrobial collection procedure conducive to both deep microbialcommunity surveys (DNA/RNA analyses), culturing and isolation of livingmicroorganisms; (2) identification of specific target microbialassociations capable of rapid transformation of subsurface organicmatter to biogas via e.g. the methylotrophic pathway, using empiricalcorrelation of microbial profiling (in alternative embodiments usingpyrosequencing) data to key geochemical parameters and targetedincubations (e.g. with lignin or other coal-analogues or precursors);(3) simultaneous identification of unfavorable, endemic microbes orconditions showing negative correlation to biogas formation using thesame information as in step #2; (4) formation characterization of bothindigenous organic carbon-rich Substrates and inorganic mineralogyaffecting the injectate-water composition for biogas formation(including core-water-microbe experiments); (5) optimization of aninjectate water chemistry (especially water pH and essential nutrients)and microbiology (selected isolates or pre-grown successful communitiesobtaining high methanogenesis rates with targeted coal and coalanalogues) to promote subsurface biogas production at the reservoirtemperature of the target field); (6) investigation and modeling ofdelivery mechanisms to successfully spread the water-soluble amendmentsand cultured microbes to the target formations; and (7) fieldimplementation of any one or all of these steps in e.g., a biogasproduction process. In other embodiments the invention may include,evaluation of the potential for biomass formation and scaleprecipitation associated with adding amendments and cultured microbes toexisting field conditions; simulation of biogas in a sub-surfacereservoir using a computational model; monitoring injected fluids,biogas, and changes in the microbial community; or field implementationof any one or all of these steps in e.g., a biogas production process.

In alternative embodiments, the compositions and methods of theinvention identify, mimic and/or manipulates combination of parametersthat result in the specificity of a methanogenic pathway in thesub-surface, including e.g., any one or a combination of parameters, forexample: i) type of organic matter (e.g. plant vs algae derived), ii)thermal maturity of organic matter (level of aromaticity and hencerecalcitrance), iii) formation water chemistry (i.e. salinity, pH,inorganic and organic water chemistry), iv) temperature, and v) presenceof appropriate syntrophic bacterial community able to provide specificmethanogenic substrates.

In alternative embodiments, the invention provides compositions, e.g.,nutrient mixes, and methods of enhancing biogenic methane productionthrough the creation of customized nutrient amendments (e.g.,supplements, mixes and the like), wherein the compositions and methodscan be used to specifically stimulate (or inhibit, as appropriate)functionally important constituents of a microbial community responsiblefor biogas formation (or responsible for inhibition of optimal biogasproduction). In alternative embodiments, the invention providesmicrobial compositions (including bioreactors) to augment microorganismsinvolved in the methanogenic degradation of recalcitrant organic matteror to introduce new microbial functionalities into a reservoir toinitiate or stimulate this process.

In alternative embodiments, the invention can identify a microbialcommunity present in a subsurface carbonaceous reservoir, e.g., bynucleic acid (e.g., DNA, RNA) characterization, e.g., by sequencing,hybridization, PCR and the like, to determine or characterize themicrobes present (optionally including their relative abundance); and inalternative embodiments a customized nutrient mixture providedcomprises, or is based on: (1) published nutrient requirement valuesthat are weighted toward the more abundant and important (relative tothe targeted methanogenic pathway) organisms; and (2), fieldobservations about specific reservoir conditions (e.g. water chemistry,well production, etc.). In alternative embodiments, the resultingcustomized nutrient composition of the invention is introduced to areservoir through an injection process at the well head, or is used in abioreactor of the invention.

In alternative embodiments, the resulting customized nutrientcomposition is used in a bioreactor, optimized through abioreactor-nutrient optimization test, and/or introduced to a reservoirthrough an injection process at the well head as required to optimizebiogas production in the bioreactor and/or in the hydrocarbon formation.

In another embodiment, the invention characterizes, e.g., by sequencing,hybridization, PCR and the like, microbial communities present in asubsurface carbonaceous reservoir. In one embodiment, a customizednutrient mixture is determined based on published nutrient requirementvalues alone that is weighted toward the more abundant and importantorganisms. The resulting customized nutrient composition used in abioreactor, optimized through a bioreactor-nutrient optimization test,and/or introduced to a reservoir through an injection, process at thewellhead as required to optimize biogas production in a bioreactorand/or in a hydrocarbon formation.

In alternative embodiments, the invention characterizes, e.g., bynucleic acid sequencing, hybridization; PCR and the like, microbialcommunities present in a subsurface carbonaceous reservoir. Theresulting customized nutrient mixture of the invention can be determinedbased on published nutrient requirement values.

In another embodiment, nutrient formulations that were developed for onereservoir are utilized for another reservoir with similar propertiessuch as geological history, geochemistry, source of carbon and microbialcommunity composition.

In alternative embodiments, the rate of methanogenesis in a subsurfacereservoir harboring coal and other recalcitrant organic carbon sourcesis increased by introduction of one or more members of a genus selectedfrom the group consisting of Methanolobus, Methanobacterium,Methanothermobacter, Methanogenium, Methanogenium, Methanofollis,Methanoculleus, Methanocorpusculum, Methanococcus, Methanocalculus,Methanobrevibacter and Methanosarcina (as pure or nearly pure culture,e.g., greater than about 70%, 80%, 90%, or 95% of cells, are from oneparticular genus) through injection at the well head. The cells may beprovided as cultures, cell pellets (such as obtained throughcentrifugation or filtration), or lyophilized preparations that arereconstituted.

In alternative embodiments, Methanolobus, Methanobacterium,Methanothermobacter, Methanogenium, Methanogenium, Methanofollis,Methanoculleus, Methanocorpusculum, Methanococcus, Methanocalculus,Methanobrevibacter and/or Methanosarcina cells (e.g., as pure,substantially pure, or nearly pure culture, e.g., greater than about50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more of cells in culture)(e.g., in a nutrient mix, a fluid, or composition, e.g., a mud) areintroduced into a subsurface reservoir or deposit, or isolated, mined orexcavated source, e.g., that has (comprises) gas, coal, oil, heavy oil,tar-sand, bitumen and the like.

In alternative embodiments, Methanolobus, Methanobacterium,Methanothermobacter, Methanogenium, Methanogenium, Methanofollis,Methanoculleus, Methanocorpusculum, Methanococcus, Methanocalculus,Methanobrevibacter and/or Methanosarcina cells, (optionally e.g., aspure, substantially pure, on nearly pure culture, e.g., greater thanabout 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more of cells inculture) are delivered to the subsurface reservoir or deposit, orisolated, mined or excavated source, through injection as enrichmentcultures, where optionally they comprise a substantial or significantportion of the total number of cells, e.g., equivalent to at least about1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% or more by cell number.

In alternative embodiments, Methanolobus, Methanobacterium,Methanothermobacter, Methanogenium, Methanogenium, Methanofollis,Methanoculleus, Methanocorpusculum, Methanococcus, Methanocalculus,Methanobrevibacter and/or Methanosarcina cells (e.g., as pure,substantially pure, or nearly pure culture, e.g., greater, than about50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more of cells, in culture)are used in a bioreactor, fluid, composition or product of manufactureof the invention.

In alternative embodiments, the invention also provides methods toenrich or select for endemic organisms capable of converting acarbonaceous material of interest that can then be re-injected into aformation to enhance methanogenesis rates, or inhibit or decreaseendemic organisms that inhibit or decrease biogas formation. In oneembodiment, this process of the invention is useful because it selectsfor the most important organisms required for the entire degradativemethanogenic pathway from a pool of organisms that are already selected(e.g., through natural selection) for growth under reservoir conditions.These methods also can be used to enrich an environment of a bioreactorof the invention.

In alternative embodiments, cells present in production water are usedto inoculate enrichment cultures containing defined medium (mineralsalts, trace metals, vitamins), where the only carbon source (abovetrace levels) is provided as the reservoir carbonaceous material and/orchemical analogues thereof. Growth of the cultures is monitored bymeasuring changes in headspace pressure (e.g., as described by Sheltonand Tiedje 1984) and methane production (e.g. using GC/FID as describedby Strapoc et al., 2008) and in increased numbers of cells present thatresults in increased turbidity. Once a significant amount of growth isdetected, the culture is passaged into fresh medium (e.g., about 1 to100-fold dilution). This procedure can be repeated indefinitely. Theseprocedures are well known to those skilled in the art and are describedin detail in general microbiology textbooks (e.g. Manual ofEnvironmental Microbiology, 3rd edn. Hurst, C. J., Crawford, R. L.,Garland, J. L., Lipson, D. A., Mills, A. L., and Stetzenbach, L. D.Washington, D.C., USA: ASM Press, pp. 1063-1071.). Prior to injectioninto the reservoir the culture can be passaged into a large capacityfermenter to produce, large number of cells. These methods also can beused to produce a bioreactor of the invention.

In alternative embodiments, cells present in production water are usedto inoculate enrichment cultures containing defined medium (mineralsalts, trace metals, vitamins), produced water, or nutrient-amendedproduced water, where the only carbon source provided is a chemicalanalogue or multiple analogues of the reservoir carbonaceous material.In this embodiment, use of tested chemical analogues allows fasterbiomass growth, e.g. prior injection into the reservoir, of the culturesthan in cultures using only the reservoir carbonaceous material. In yetanother embodiment, cells present in production water are used toinoculate enrichment cultures containing defined medium supplementedwith a customized nutrient mix where the only carbon source (above tracelevels) is provided as the reservoir carbonaceous material. The cellscan be inoculated into enrichment cultures of a bioreactor of theinvention.

In alternative embodiments, cells isolated from or microbial consortiafound in other formations, basins or environments are used to inoculateenrichment cultures containing defined medium or target produced watersupplemented with a customized nutrient mix where the only carbon source(above trace levels) is provided as the target reservoir carbonaceousmaterial or analogue of thereof or carbonaceous material from otherreservoir or basin. The cells can be inoculated into enrichment culturesof a bioreactor of the invention or the target reservoir or otherreservoir, or basin.

In alternative embodiments, the cells produced from the aforementionedenrichments or fermenter are lyophilized for storage and transport tothe well site where they are mixed with water and customized nutrientformulations immediately prior to injection. The cells would belyophilized in the presence of reducing agents to protect themethanogens and other obligate anaerobes from oxidation during storageand injection. These cells also can be inoculated into enrichmentcultures of a bioreactor of the invention.

In alternative embodiments the invention provides methods for analysisand understanding of subsurface microbial communities responsible forconversion of coal and coal-like substrates into methane, and forcontrolling geochemical conditions. Thus, in alternative embodiments,the invention, compositions and methods of the invention are used tostimulate preferred subsurface methanogenesis pathways and to enhancethe rates of biogas formation.

In alternative embodiments, compositions and methods of the inventionsupply deficient nutrients (e.g., enhanced metal salts of compoundsfound in methylotrophic/bacterial enzymes, non-inhibitory level ofalternate electron acceptors such as iron, manganese, or other nutrientsand trace elements identified by correlating nutrient abundance tomicrobial growth/methane production) and/or modifying some parameters ofthe formation water (e.g., higher pH to the optimal range of themicrobial association from culture experiments at the reservoirtemperature) can shift microbial populations of all wells towards moreefficient coal/kerogen biodegrading (e.g., in Belugamethanol/methyl-generating) and increase the methanogenesis rates.

In alternative embodiments, compositions and methods of the inventioncomprise use of subsurface exploitation of methylotrophic (e.g.,methanol and other methyl-containing) substrates under neutral toslightly alkaline conditions to enhanced biogas formation.

In alternative embodiments, methods of the invention identify: (a) keyand fastest operating microbial pathway for subsurface biogas formationand (b) ranges of key environmental geochemical parameters thatstimulate this pathway thus enabling a means to optimize subsurfacebiogas-production rates and generate a positive offset to the gasfield's production decline. In alternative embodiments, carefulstimulation of the subsurface bioreactor (by inspecting and adjustingchemistry and microbiology of co-produced and re-injected water) ensurespotential long term stable methane production rate (10's of years),owing to vastness of accessible organic matter (organic debris andbedded coals) in the subsurface.

In alternative embodiments, the invention provides a specific integratedprocess for microbe discovery (including syntrophic associations betweenorganic-matter degrading bacteria and gas-producing Archaea) and anoptimization strategy for developing a supplemental water injectatepromoting biogas growth and minimizing deleterious effects.

In alternative embodiments, the invention can assess the formation ofcarbon mass and characterize the geochemical bio-convertibility oforganic matter, and follow-up enrichment 10 experiments on indigenousformations required for potentially successful field implementation.Amendments can be extremely cost-effective.

In alternative embodiments, compositions and methods provided can bepracticed to enhance and produce natural gases from certain Cook Inletfields which contain biogenic methane almost exclusively. In alternativeembodiments, compositions and methods provided can be used to enhancemicrobial communities that are still active at present day in both theBeluga and Sterling formations, degrading complex organic matter tosimpler compounds that in turn can be biologically transformed tomethane. Thin coals and dispersed organic debris in the sand-dominatedfluvial system are easily accessible for microbial attack. Faster ratesof biodegradation and methanogenesis can be achieved by selecting forspecific microbial populations through adjusting the chemistry offormation waters (i.e. pH, Eh, as well as trace elements and nutrientssuch as Mo, Ni, phosphate, ammonia, etc.).

Several parameters of Cook Inlet microbial communities including 16SrRNA gene profiling, metagenomics analysis, cultivation screens andgeochemical analysis were studied in the lab for potential future fieldimplementation. Both the Beluga and Sterling formations are excellentcandidates for a field pilot for enhancement of microbial methanegeneration. These formations have low reservoir temperatures,association of organic matter within and adjacent to highly porous andpermeable sands with organic debris and nutritious volcanic ash, andreasonably good lateral connectivity within the Sterling formationreservoirs.

DNA, culturing, and geochemistry of Cook Inlet microbial associationswere studied in the lab for potential future field implementation. Boththe Beluga and Sterling formations are excellent candidates for a fieldpilot for enhancement of biogas These formations have low reservoirtemperatures, association of organic matter within and adjacent tohighly porous and permeable sands with organic debris and nutritiousvolcanic ash, and reasonably good lateral connectivity within theSterling formation reservoirs.

In alternative embodiments, the term “carbonaceous” is defined as anyrock containing organic carbon (carbonaceous rocks such as coal, shale,sandstone or limestone with organic debris or oil) with a total organiccarbon (TOC) content >0.5 weight % (wt. %).

In alternative embodiments, the term “coal” is defined as a readilycombustible rock containing 50 wt. % TOC.

In alternative embodiments, the term “correlation” is defined as therelationship or degree of similarity between two variables. Correlationanalyses may be performed by any method or calculation known in the art.Correlation analyses for R and R₂ may be performed as described by M. J.Schmidt in Understanding and Using Statistics, 1975 (D.C. Health andCompany), pages 131-147. The degree of correlation for R is defined asfollows:

1.0 Perfect  0.7-0.99 High 0.5-0.7 Moderate 0.3-0.5 Low 0.1-0.3Negligible

In alternative embodiments, the term “field observation” is defined asthe set of reservoir parameters that include: gas composition andisotopes, water chemistry, pH, salinity, Eh (redox potential),temperature, depth, production parameters and history, description andcharacterization of the formation, e.g. description, sampling oranalyses of core, cuttings or outcrop rock material.

In alternative embodiments, the term “production water” is defined aswater recovered co-produced with any petroleum or hydrocarbon productsat the well head.

In alternative embodiments, the term “recalcitrant organic matter” isdefined as any organic matter that is generally resistant tobiodegradation by the action of microorganisms, e.g. highly aromaticcoals.

In alternative embodiments, the term “chemical analogue” is defines asspecific chemical compound or compounds of structure and bond typesrepresentative of the target carbonaceous material. Such chemicallydefined analogue has known chemical structure, is commercially availableand can be used as a surrogate for faster growth of targeted consortium.

In alternative embodiments, biogenic gas formation is modeled in one ormore subsurface formations. Biogenic gas formation modeling includesdetermining changes in the formation composition and gas formation asbiogenic growth occurs. Modeling includes estimating changes in organicmatter content in the formation, volume of gas generated during biogenicgrowth, and determination of potential gas flow paths through theformation and travel time of biogas from biogenesis to production, basedon a geological characterization and model of the formation.

In alternative embodiments, careful sample collection of gases and theco-produced water at the well-head improved identification of microbialcommunities associated with potentially commercial geochemical processesand was facilitated by proper treatment of water samples to preservemicrobes and water chemistry during transit to and storage at thelaboratory. About 1 L of non-filtered water was collected for DNAextraction and 16S rRNA gene profiling using pyrosequencing. Additionalwater samples were collected in 160 mL serum bottles for enrichments(amended with resazurin and sodium sulfide with blue butyl stoppers).Another 1.5 L of water was filtered on-site using 0.22 μm pore sizefilters. Filtered water samples were split for variety of subsequentanalyses including inorganic (cations—fixed with HCl, anions), andorganic chemistry (volatile fatty acids—amended with benzalkoniumchloride, alcohols). Well-head gas samples were taken for molecular andisotopic composition of the gas using e.g., ISOTUBES™ (IsoTech). Inaddition, the field pH, Eh, salinity, temperature of the waters,alkalinity (via titration), and/or other properties were measured assoon as possible after water collection. In alternative embodiments, anintegrated (wide and deep) screening of both geochemical andmicrobiological environmental properties was used to characterizesubsurface microbial environments; thus, providing ah accuratebackground for the composition of the reservoir.

In another embodiment, genomic DNA is extracted from samples of asubsurface carbonaceous reservoir of interest. Genomic DNA may beextracted from the samples by any of a number of commercially availablekits (e.g., POWERSOIL™ available from MoBio Laboratories Inc. (Carlsbad,Calif.) or FASTDNA™ kit by Q Biogene) or by methods ordinarily known bythose skilled in the art of environmental microbiology. The microbialcommunities resident in the reservoir samples are profiled (orinventoried) by determining the DNA sequence of a portion of the 16SrRNA genes present. This gene is widely used as an indicator, or barcodefor a given microbial species (Pace, 1997). The 16S rRNA genes arerecovered from genomic DNA through PCR amplification using primers thatare designed to conserved regions with the gene. Such primers are wellknown in the art. For example, the primers TX9 and 1391R (e.g., seeAshby, Rine et al. 2007, see list below) amplify an approximately 600base-pair region of the 16S rRNA gene that includes the fifth througheighth variable (V5-V8) regions. The DNA sequence of the resulting 16SrRNA amplicon may be determined using any available technologyincluding, but not limited to, Sanger, or ‘next generation’ technologiessuch as those available from Roche, ABI, Illumina, Ion Torrent orPacific Biosciences. Determination of the number of times each sequenceoccurs in a sample provides an indication of the microbial communitystructure (e.g. see Ashby, Rine et al. 2007). The abundance of eachsequence identified from a given sample can be compared with that ofevery other sequence to identify sequences that show significantcorrelations to one another. These sequences are likely to be members ofthe same consortium and participate in common biogeochemical process(Ashby 2003). The microbial communities may also be characterized bysequencing genes other than the 16S rRNA genes or even by random shotgunsequencing of genomic fragments by methods that are well known in theart, (Venter, Remington et al. 2004). The microbial communities may alsobe characterized by cultivation dependent approaches that are well knownin the art. For example, this approach, may identify organisms throughmetabolic capabilities, morphological considerations and Gram stains.Total count of microbial sequences in the Cook Inlet gas field wasdominated by Methanolobus (FIG. 1).

In one embodiment, specific target microbial Bacteria-Archaeaassociations favorable for biogas production are identified through anintegrated water and gas-sampling strategy that allowed for the searchacross biological and geochemical parameters for environmentalcorrelations between microbe associations and key chemical processeswith potential commercial value. For the purposes of high-gradingenhanced biomethane production, the correlations have been based on twospecific microbial enrichments of the Cook Inlet formation waters with:(a) common methanogenic substrates (combination of CO₂/H₂, acetate,methanol substrates) to gauge the general health of the endemicmethanogenic community and (b) with lignin/lignin monomers-supplementedCook Inlet coals/organic matter-rich sandstone enrichments to simulatefurther bacterial breakdown of organic macromolecules to specificsubstrates vital to the growth of key Archaeal methanogens. Statisticalcorrelation of geochemical data from the formation water and themicrobial distribution data (expressed as Z score values oflog-transformed 16S rRNA gene sequence occurrence data), hassuccessfully identified microbial associations and potential syntrophiesas well as their affiliation to specific ranges of geochemicalparameters (i.e. pH, salinity, temperature, trace metals, gas isotopiccomposition). Sequence occurrence and geochemistry data from multiplewells and/or basins can be used. For the Cook Inlet gas fields, 16S rRNAgene pyrosequencing data integrated with these two biogas-productiondatasets clearly show that methanol and other methyl-containing speciesare the most efficient substrates for biogas formation via themethylotrophic pathway. The highest methane production rate correspondedto highest formation-water pH and was dominated by methanol/methylutilizing genus Methanolobus (FIG. 2). The correlations of 16S rRNAsequence data achieved with new generation 454-sequencer also pointedput specific microbial associations potential syntrophies betweendifferent microbial groups. For example, Family Methanosarcinaceae(Class Methanomicrobia) is capable of utilizing methyl-containingcompounds (i.e. methylamines) as substrates for methanogenesis and themain Cook Inlet methanogens belong to this family: Methanolobus andMethanosarcina.

Lab experiments were able to determine the ability of microbial culturesor microbes present in produced water to convert different types oforganic matter (OM) to methane: (a) subsurface OM (2 types of coal, (b)coal-mimicking substrates (i.e. lignin mix, 0.4 mL/L*d, yield up to 15%mass to mass), and (c) biowaste materials (i.e. refinery sludge,biocoals, up to 0.9 mL/L*d). Furthermore, molybdenum (Mo) (goodcorrelation with methanogenesis rate), nickel (Ni), tungsten (W),phosphate and ammonia were considered as important nutrients.

Additionally, the methylotrophic biogas formation correlated withneutral to slightly alkaline conditions in the formation waters (FIG. 2,pH greater than 7.2 with an optimum approximately 7.5). Methanogenesisrate is measured using a pressure transducer and GC/FID. Alternatively,rates of intermediate steps can be measured, by inhibiting methanogens(i.e. with BESA) and analyzing the enrichment water chemistry includingvolatile fatty acids (VFA's), alcohols and the like. Similarly,substrate material (i.e. coal, oil-sands, bitumen, and the like) can becharacterized before and after enrichment (i.e. conversion to methane)for chemical structure (i.e. NMR, FTIR). The bacterial break-downpolymers of macromolecular subsurface OM (the rate limiting step) can bealso enriched by using: (a) potential synthetic syntrophic microbialassociations inferred from this research or (b) by amending anenrichment of indigenous microbial populations (i.e. on coal or coalanalogues).

In another embodiment, unfavorable endemic bacteria or environmentalconditions affecting biogas formation were identified. Endemic bacteriathat did not produce methane, environment unfavorable to endemicmicrobes that did produce methane, or conditions that show a negativecorrelation to biogas (e.g., methane) formation were identified. Theintegrated data from microbial DNA, geochemistry, and biogas productionvia enrichment experiments are also used to find negative correlations,indicating possible specific microbes or environmental conditionsdeleterious to biogas formation. Negative correlation within DNAsequences and against geochemistry are also taken into account aspotential microbial rivalry/inhibition and toxicity/unfavorable chemicalconditions (i.e. high propionate concentration, large populations ofnitrate or sulfate reducing bacteria, typically inhibiting methanogens),respectively. Potential risk of fouling of the bioreactor includingproduction hydrogen sulfide and accumulation non-reactive acidicproducts is an important element of the targeted injectate-water recipe,such that biogas formation by the methylotrophic pathway is optimizedfor their essential growth substrates without impeding production due toother factors. For example, microbial populations derived from the CookInlet subsurface waters are also tested for the extent of microbialremoval of individual VFAs and retardation of microbial pathways leadingto potential VFA buildup (and lowered pH to unfavorable acidicconditions). Microbial populations from most of the wells were capableof stoichiometric conversion of butyrate and acetate to CH₄ within a fewmonths. In contrast, propionate was not degraded in any of the samplesand its buildup in a subsurface bioreactor has a likely deleteriousimpact on biogas production by the methylotrophic pathway. Therefore, insome embodiments, potential stimulation of propionate generation viasupplemental injectate-water must be avoided, in addition, theintroduction of certain anions such as sulfate and nitrate is to beavoided.

In one embodiment, injection zone placement and injectate watercomposition were determined based on formation characterization inorganic carbon-rich formations and through inorganic mineralogy. Thecarbonaceous substrate is as important as the microbial community inachieving biogas formation at economically significant rates. Our workshows a relationship between biogas rate and substrate thermal maturity(measured by vitrinite reflectance or another geochemical parameterexpressed in vitrinite-reflectance equivalence). Furthermore, theformations targeted for stimulated biogas growth must have sufficientorganic mass, contain microbial enzyme-accessible chemical-bond types,and also allow for fluid injectability at sufficiently meaningful rates.Thus, in alternative embodiments, methods of the invention can comprisegeochemical characterization of: (a) the mineralogy (e.g., content ofthe nutritious volcanic ash clays using XRD, their chemical compositionand ion exchange potential using SEM/EDS, association with organicmatter particles using thin sections and SEM), (b) organic matter(functional groups and bond type distribution using NMR, TOC, ROCK-EVAL™pyrolysis, organic petrography including vitrinite reflectance and OMfluorescence), and (c) correlation of organic-content of core samples towell-logs for biogas resourcing, and formation-evaluation of fluid flow(porosity, permeability, swelling). In addition, clays and otherminerals within the organic matter-rich formations can be studied forion exchange. Potential interactions between any proposed injectate andindigenous formation water can be evaluated using advancedphysical-chemical and transport modeling.

In another embodiment, injectate water chemistry is optimized for biogasproduction enhancement. Methods of the invention comprise use ofgeochemical correlations with desired microbial associations to optimizebiogas-formation rate/yield by chemistry adjustment; this information isused to make a injectate-water recipe used to practice this invention,including the use of pH buffers. The highest biogas-formation on anideal substrate medium (combination of CO₂/H₂, acetate, methanol) andhighest biogas production rate on a lignin/lignin monomers-supplementedCook Inlet coal-lignin enrichment has been achieved by themethyl/methanol-pathway associated microbial community derivedexclusively from wells with pH>7.23 (FIG. 2), strongly implying higherpH as an alternative condition (neutral-slightly alkaline) and animportant basic makeup of injectate-water recipes of this invention.Thus, in another embodiment, compositions and methods comprise use ofrelatively alkaline (high) pH nutrients, injectate-water, liquid recipesand the like, and use of buffers biasing an alkaline (high) pH. Inalternative embodiments, injectate water/may also includesupplementation with the best performing microbes on target substrate orits chemical analogue, even if derived from different environment (e.g.another oil or CBM basin), that performed well in the enrichments. Forthe targeted fields, the indigenous mineralogy, organic matter, porositystructure are likely to affect the growth of methanogenic microbes, andin some cases, microbial biofilm (e.g., via surface adhesion and miningnutritious mineralogy due to the presence of clays, volcanic ash, and/ororganic debris) under the supplemented water conditions. Therefore, thetargeted microbial community and favorable environment (e.g., optimizedpH, supplemental macro- and micro-nutrients, vitamins) may be adjustedfor interactions with the native organic-containing formations (FIG. 6).In addition, undesirable dissolution or precipitation of mineral phasespotentially harmful to the microbes or the reservoir quality can beassessed; e.g. tested using chemical modeling, PHREEQC—mineralsolubility changes caused by interactions of formation water andminerals with the injectate. A minimalistic approach can be used tofavorably enhance the targeted biogas-forming bacterial-archaealmicrobial association, while not promoting over-enhanced growth of othermicrobes not important to the biogas formation process (i.e., to avoidwater injection delivery problems due to biofilm plugging around wellinjectors).

In one embodiment, customized nutrient amendments are provided. Anutrient mix customized for a specific microbial community, which e.g.,can be developed by the following steps (FIGS. 4 and 5):

1. Transform microbial 16S rRNA gene sequence count data for all samplesincluding: adding a small value to each sequence count (e.g. add 1/10thof the lowest value observed to every sequence count, thus avoidingtaking log of zero; log 20 transform sequence counts and determineZ-scores (for a given sequence in a given well, by subtracting the meanvalue of the occurrence of a given sequence in all wells examined anddividing the resulting number by the standard deviation of the samearray)

2. Determine correlation of the distribution of all sequences todistribution of target sequences, e.g. dominant methanogens responsiblefor leading methanogenic pathway (tested experimentally withcoal/lignin/lignin monomers incubations, e.g. dominating Methanolobussequence using transformed data) to obtain Pearson correlationcoefficients Ra and Rb.

3. Sort sequences based on their R values. Select sequences with Rhigher than cut off value (e.g. 0.70). Subsequently, remove sequenceswith low counts (e.g. 300) with small contribution to the community andhaving the sequence count in a range of potential sequencing error.

4. Remaining sequences form so called Consortium, consisting of bacteria(b) and Archaea (a) related or similarly distributed to the selecteddominant methanogen. Correlations to sum of several grouped sequences(e.g. syntrophic microorganisms) can be also used.

5. 16S rRNA gene sequences in the Consortium are identified bycomparison to annotated DNA sequence database (e.g. NCBI).

6. Nutrient and growth condition (e.g. pH, Cl−, NH4+, etc.) requirementsfor of each of the selected microbial genus/or microbial strains wasdetermined using information from publicly available literatures and incertain cases using information from the German Resource Center forBiological Materials. From this, an optimal Recipe Concentration (CR) ofeach element (e.g. Mg) or condition (e.g. pH) X for each Consortiummember (a or b) was obtained.

7. Final Recipe Concentration (C_(FR)) of given element or condition (X)for entire Consortium is obtained by using following equation (1):

$\begin{matrix}{C_{{FR},X} = {{f_{B} \times {\sum\limits_{n}{C_{R,X,{bn}} \times f_{bn} \times r_{bn}}}} + {f_{A} \times {\sum\limits_{m}{C_{R,X,{am}} \times f_{am}r_{am}}}}}} & (1)\end{matrix}$

-   -   Where C_(FR,X) is the final recipe concentration of element X        for a value or condition X, e.g. pH; C_(R,X,br;) is a        literature-based recipe concentration of element or condition X;        f_(B) and f_(A) are optional weighting parameters for bacteria        vs Archaea in a population of targeted well, formation, and/or        incubation conditions; f_(bm) is the fraction of bacterial        sequence n out of total bacterial sequence count within the        consortium; f_(am) is the fraction of archaeal sequence m out of        total archaeal sequence count within the consortium; r_(bn) is        the Pearson correlation coefficient of a bacterial sequence n to        selected dominant sequence (e.g. main methanogen) or grouped        sequences (e.g. syntrophic association); and r_(am) is the        Pearson correlation coefficient of an archaeal sequence m to        selected dominant sequence (e.g. main methanogen) or grouped        sequences (e.g. syntrophic association). If conditions for        Archaea and bacteria are equal both f_(A) and f_(B) parameters        are equal to 0.5. If however, Archaea or bacteria are dominant        in a formation or injectate, or if Archaea or bacteria are more,        critical or rate limiting, f_(A) or f_(B) can be adjusted to        account for differences in consortium population, overall        activity, or other factors dependent upon the specific process        or conditions in the targeted well, formation; and/or        incubation.

8. Final calculated C_(FR) values contribute to the final recipe (FR).

9. Additional minor adjustments of the calculated parameters are testedone parameter at a time while maintaining other parameters. In oneembodiment changes are assessed in a sand-pack bioreactor (Table 1, FIG.7).

10. Subtract all amendments (X) present in the formation water (FW) toobtain ah adjusted, final recipe (AFR) for the current well conditions(FIG. 6).

11. Small adjustments to the AFR may be made to accommodate chargebalance, increase chemical stability, and ensure no precipitation occursduring mixing, storage, injection or under formation conditions.Chemical stability, may be calculated using PHREEQC™, PHREEQCT™, orPHAST software from USGS and others, AQUACHEM™ from Waterloo, Inc.,ROCKWARE™, as well as other programs are also available to analyze watersalinity and precipitation under various conditions.

The nutrient composition may be introduced to the reservoir throughinjection of a single bolus, through a continuous (e.g., a bleed in)process, or a pulsed process. The AFR may be amended dependent upon thechanges in the production water, methane production, and/or microbialcomposition over time. The same methods used to re-inject produced waterinto a well may be used to inject/re-inject a mixture of produced waterand nutrient concentrate.

In another embodiment, the final water injectate is delivered to thetarget formation to induce or increase biogas production. Fieldimplementation, delivery of the designed injectate, may be improvedwhere good well-to-well connectivity exists through highly permeablecontinuous formation intervals. Core description, geophysical logs, andpermeability/porosity data may be used to identify target wells,optimize injection intervals, and improve biogenic gas production. Forsandstones containing dispersed organic debris, injection ofsupplemented water may be applied to the: (a) near-water leg or (b)previously depleted gas-bearing zones, in the same formation down-dipfrom the free-gas zone (FIG. 11). Consequently, new microbial gas formedin the water leg migrates upwards, to the gas column, supplementing theoverall gas reserves. Nevertheless, converting these large resources ofsub-surface organic matter require that the injectate-water supplementcontact a large volume, of the formation, without choking off injectionaround the well-bore due to biofilm growth. Therefore, an importantconsideration prior to implementation is investigation of the use oftime-release substances or near-well toxic concentrations to preventbiofilm plugging, followed up by bench testing on target formations. Inan alternative embodiment, the methods involve continuous injection ofnutrients at final concentration (i.e. bleed in of concentratednutrients into produced water-disposal well). Another option is deliveryof nutrients with the fracturing fluids used often during completion orre-completion of gas producing wells.

In alternative embodiments, tracing injected water migration, biogasformation and changes in microbial communities are critical tobenchmarking success. Water migration can be traced using water solublegeochemical tracers (i.e. stable or radio isotopically labeled ions suchas ¹³C or ¹⁴C carbonate and ¹²⁹iodine or ³⁶ chlorine, bromide). Newlygenerated biogas can be traced from gas isotopes, using ¹⁴C, ¹³C, ²H or³H enriched methanogenic substrates, including bicarbonate, lignin andaromatic monomers. Additionally, production profile of nearby producingwells can be observed together with gas to water ratio, gas pressure,production rates, and gas dryness. Biomass tracers of newly grownmicrobes can be also used, including ¹⁴C, ¹³C, ²H or ³H-labeled organiccompounds (i.e. lignin monomers, DNA, amino acids, bacteriophage, orother coal analogues, i.e. aromatic substrates listed in Example 4),¹⁴N-enriched ammonia. Monitoring will also include microbial communitychanges through RNA/DNA profiling, RNA/DNA yields.

In another embodiment, chemical analogs of Subsurface organicmatter-containing rocks allow for quick growth of biomass in microbialconsortia that can be re-injected, e.g. when the optimized nutrient mixis identified. For low thermal maturity coals, a lignin monomer mixturehas been used to benchmark high methane producing consortia capable ofthe critical depolymerization step (FIG. 3). Coal depolymerization isthought to be rate limiting in the coal biogasification process. Forhigher maturity coals (about 0.6 to 1.4% R₀) aromatic analogs are testedas surrogates for biogas formation, including biphenyl 4-methanol,methoxy biphenyl 1,1,-biphenyl, methyl, dimethyl, phenanthrene, andother compounds that mimic degraded coal monomers.

In one embodiment, a biogenic gas formation is assessed by developing afacies model, determining formation parameters and distributing theseparameters for each facies in a geocellular model. This geocellularmodel can then be used to simulate and history match any previousgas/water production to validate the model, and then to simulate futurebiogenic gas production with nutrient optimization. As biogenic gasproduction continues, the initial model may be updated based on currentproduction trends with optimized nutrient formulations. One or moregeocellular models may be developed using numerous formation modelingtechniques including ECLIPSE™, GEOFINDIQ™ from Schlumberger, MPS(Multiple-Point Statistics), FDM (Facies Distribution Modeling) andother geocellular modeling techniques and programs; including techniquesand tools developed in house or by independent programmers. Formationparameters can include % TOC (total organic carbon), density, porosity,permeability, pressure distribution, saturation (gas, water, fluid, oil,etc.), conductivity, impedance, continuity, flow rate or velocity,volume, compressibility, thickness, fluid viscosity, and otherproperties. Formation parameters may be measured directly or indirectlythrough well logging, measured through core samples and analysis, orestimated based on various formation properties. Formation propertiesmay be distributed by estimating from one sample well to the next,interpolating, or applied by simulation algorithms including Kriging,stochastic simulation, Markov Chain Monte Carlo distribution, and thelike, as well as combinations of these methods. Biogenic gas productioncan be simulated using STARS™ from Computer Modeling Group, Ltd.,JEWELSUITE™ from Baker Huges, BASINMOD™ from Platte River, or otherreservoir simulation software as well as programs designed and developedin house or by independent programmers. Some software may incorporateboth geocellular modeling and reservoir simulation.

In another embodiment, a geocellular model is developed as describedabove and used to test gas flow, travel time, and continuity of thereservoir against variabilities in key formation parameters, which maybe the result of limited or conflicting geological data. Once these keyparameter variabilities are identified, the reservoir analysis may besimplified. In one embodiment, the travel time for biogenic gas in thereservoir was determined by modeling the biogenic gas production rate asactual gas injection into an injection well. This allowed for multiplevariations of the key parameters in the geo cellular model to besimulated significantly faster than would be possible with a reservoirmodel which included the full biogenic gas production. This quick methodhelped to define the possible range in gas travel time based onvariabilities in formation parameters and to identify which formationparameters are most influential on gas travel time and require furtherinvestigation to narrow their variability.

In another embodiment, risk analysis is used to identify potentialrisks, evaluate risk severity and probability, propose possiblemitigation strategies, design tests for each risk, and test each riskand putative preventive action. Potential risks associated with biogenicgas production can be identified from product suppliers, through wateracquisition, to nutrient injection, to microbe growth and biofilmformation, in one embodiment, potential risks include impurities in oneor more of the nutrients, additives, treatments, water, or otherfeedstreams; contamination with oxygen, sulfur, or other compounds;contamination with one or more microbes; scaling in the injection line,wellbore and/or formation; biofilm formation in the mixing tank, storagetank, injection line, wellbore and/or formation; sludge formation in themixing tank and/or storage tank; biocorrosion in the mixing tank,storage tank, and/or injection line; formation of hydrogen sulfide(H₂S); oxygen removal; biomass plugging; and the like, eitherindividually or in conjunction with other risks. Some risks maycontribute to or correlate with other risks, for example sludgeformation in the storage tank and biofilm formation in the injectionline may both be the related to increased biomass in the storage tank.

Additionally, in some embodiments, enrichment of bacterial culturesusing analogues to target subsurface organic material including ligninand lignin monomers, soluble hydrocarbons, other soluble substrates thatmimic the composition of the hydrocarbon formation are used to enhancemicrobial growth in vitro. Modeling components of the hydrocarbonformation using simple monomers identified in produced water and/orthrough decomposition of formation samples provides a ready source ofsoluble substrates for microbial growth and selection assays. Thisinnovative approach to rapid microbial growth and selection allowsdevelopment of chemical and microbial optimized amendments for thetargeted methanogenic pathway and for enhanced methanogenesis rate.Amendments were tested under current field conditions developed toevaluate the potential for biomass formation and scale precipitationresulting from the addition of amendments, which could createoperational problems in the gas production and water injectionfacilities in the field. Implementation of any enhanced biogas producingprocess must include the combined optimization of the reward (biogasformation) versus the risk factors (deleterious effects to overall gasproduction, corrosion/scaling, or gas quality).

The invention provides kits comprising compositions and methods of theinvention, including instructions for use thereof. In alternativeembodiments, the invention provides kits comprising a composition (e.g.,a nutrient composition), product of manufacture (e.g., a bioreactor), ormixture (e.g., a nutrient mixture) or culture of cells of the invention;wherein optionally the kit further comprises instructions for practicinga method of the invention.

The following examples of certain embodiments of the invention aregiven. Each example is provided by way of explanation of the invention,one of many embodiments of the invention, and the following examplesshould not be read to limit, or define, the scope of the invention.

EXAMPLES Example 1 Identification of Methanolobus spp. for MethanogenicDegradation of Coal and Other Recalcitrant Organic Matter

This Example describes the identification of Methanolobus spp. as majorcontributor to methanogenic degradation of coal and other recalcitrantorganic matter in subsurface gas reservoirs.

Production water was collected from the separator unit at the well headfrom a number of gas wells from the Beluga River Unit on the Cook Inletof Alaska. A portion of the water sample designated for microbiologicalanalysis was maintained anaerobically in sterile containers supplementedwith cysteine and resazurin (redox indicator) under an argon headspace.The samples were shipped cold to Taxon's facility in California byexpress delivery.

Genomic DNA was isolated from cell pellets obtained by centrifuging theproduction water at 4,000×g for 30 min at 4° C. The pellets wereresuspended in phosphate buffer and transferred to bead beating tubes.Total genomic DNA was extracted by a bead beating procedure as described(Ashby, Rine et al. 2007). A portion of the 16S rRNA genes werePCR-amplified using the primers TX9/1391r, followed by agarosepurification of the amplicons. The amplicons were amplified a secondtime using fusion primers to incorporate the A and B adapters inaddition to barcodes that enabled multiplexing of samples into a singlerun.

The 16S rRNA gene amplicons were sequenced on a Roche 454™ sequencerusing Titanium chemistry and standard shotgun sequencing kits followingthe manufacturer's protocol. Profiles were created by documenting thenumber of times each unique sequence occurred in each sample. Sequencescorresponding to those of Methanolobus spp. were observed to be dominantmembers of the Cook Inlet subsurface communities (sec FIG. 1).Annotation of the sequences was performed by BLAST comparisons(Altschul, Madden et al. 1997) to the Genbank database.

Example 2 Stimulation of Methanogenic Degradation of Coal andRecalcitrant Organic Matter in Model Sandpack Bioreactors by AddingCustomized Nutrient Amendments of the Invention

In order to create a culture condition that approximated the rock matrixfound in the sub-surface gas reservoir, carbonaceous material recoveredfrom core samples were mixed in the natural in situ ratios with sand inanaerobic tubes fitted with solid rubber stoppers that were crimpsealed.

Specifically, ASTM grade sand (17.8 g, U.S. Silica Company) was addedinto 15 ml polypropylene conical tubes which was followed by addition ofcarbonaceous materials (0.167 g each of coal, sandstone with organicdebris, and volcanic ash, to represent Cook Inlet gas-bearing formation)that are derived from Miocene aged rocks (i.e. core samples) from theBeluga gas field in Alaska, U.S.A. Conical tubes representing controlset-up was amended with 22.88 g of sand but without adding thecarbonaceous materials. The mixture in the conical tubes was homogenizedfor 10 s with a vortex mixer, and all the materials above includingseveral aliquot of sterile 3.5 g sands were transferred into ananaerobic chamber that has been equilibrated with an atmosphere ofhydrogen, carbon dioxide and nitrogen (5:5:90% v/v, respectively). Then,the caps on the conical tubes were loosely opened in order to createanaerobic condition in the tubes. All experimental procedures from thispoint were carried out inside the anaerobic chamber.

After 24 h the mixture in the conical tubes that contained sand andcarbonaceous materials was transferred into sterile glass tubes (15 mL)that was previously stored in the anaerobic chamber. This mixture ofsand and carbonaceous materials was then overlaid with 3.5 g aliquot ofsand to create a ˜1 cm upper layer that is free of carbonaceousmaterials. The carbon-free mixture in the control conical tubes was alsodecanted into sterile test tubes but in this case it was not overlaidwith another layer of sand The orifice of each of the tube was cappedwith sterile stoppers and crimped-sealed with metal caps. All the tubesand its content were autoclaved for 20 min at 120.

The conditions for the experimental investigation are stated below:

-   -   i. Sand+unamended produced water from the targeted gas field    -   ii. Sand/carbonaceous materials (organic rich rocks from        targeted formations)+unamended produced water (FIG. 8)    -   iii. Sand/carbonaceous materials+standard nutrient amended        produced water based on lab experiments (1st order), and        literature, search (2nd order) as shown in FIG. 5, Step 5        (results of sand pack experiments shown on FIG. 8)    -   iv. Sand/carbonaceous materials+standard nutrient amended        produced water (FIG. 5, step 5) in which specific nutrient        parameters were varied individually or in groups    -   v. Sand/carbonaceous materials+coal/lignin-grown        enrichments+optimized nutrient amended produced water (FIG. 5,        Step 11);

TABLE 1 Stock solutions used to prepare media for sand pack experiments.Mineral nutrients Trace Metal Solution^(a) Vitamins solution^(b) (mg/l)(mg/l) (mg/l) NaCl/KCl, 5844/7455 Na-nitrilotriacetate, 1500p-Aminobenzoate, 50 Na₂SO₄•10H₂O FeCl₂•4H₂O, 200 Biotin, 20 NH₄Cl, 29.99MnCl₂•4H₂O, 100 Cyanocobalamin, 5 MgCl₂, 5.71 NaWO₄•2H₂O, 20 Folic, 20Na₂HPO₄/NaH₂PO₄, CoCl₂•6H₂O, 100 Lipoic acid, 50 55.36/46.79 ZnCl₂, 50Nicotinic acid, 50 CuCl₂•2H₂O, 2 Pyridoxine-HCl, 100 H₃BO₃, 5Thiamine-HCl, 50 NaMoO₄•2H₂O, 10 Riboflavin, 50 Na₂SeO₃, 17Ca-Pantothenic acid, NiCl₂•6H₂O, 24 50 ^(a)Roh et al., 2006 ^(b)Zinder,S.H., Techniques in Microbial Ecology, p113-134

Stock solutions listed in Table 1 were used to prepare set of solutionsfor nutrient additions to the sand pack experiments with varyingnutrient concentrations. The final optimized nutrient concentrations(mM) and the amount of other constituents in the standard optimizednutrient amended produced water (final pH 7.5) was: NaCl—KCl, 100 mM;NH⁴⁺, 5.6 mM; PO₄, 7.8 mM; Mg²⁺, 2 mM; SO₄ ²⁻, 0; 1 ml Trace elementsolution, 1×; 1 ml vitamin solution, 1× (Table 1). The pH andconcentration of NaCl—KCl, NH⁴⁺, PO₄, Mg²⁺, SO₄ ²⁻, trace elementsolution, and vitamin solution, 1×. The tubes with produced water withlignin/coal-grown enrichment were separately prepared by addition ofconcentrated stock culture of cells, which were stored frozen at −80° C.and thawed prior to use.

The final volume of nutrient and/or cell amended produced water and theunamended produced water in all the tubes (i.e. tubes with sand andcarbonaceous materials, and tubes with sand only) was 20 ml. Alltransfers into the tubes was done using a 20 gauge-6 inches BD spinalneedle which allow the transfer of the mixtures at the bottom of thetube which allowed good distribution of the equilibrated mixturethroughout the sand-packed tubes. All experimental and controlsand-packed tubes were prepared in triplicates, and the tubes werecapped with freshly prepared sterile 20 mm septum stopper which werecrimped-sealed. The atmosphere, in the headspace of all the tubes wasreplaced N2 (100% v/v) and they were immediately incubated at 20° C.

TABLE 2 Matrix of parameter variability BLANK 0 1 2 3 4 5 1 pH 7 .5 .5.5 2 NaCl/KCl 0 5 00 00 00 00 3 SO₄ 0 .1 .3 .3 4 NH₄ 0 .6 6.7 0 5 PO₄ 0.6 .8 0 6 Mg 0 0 7 Trace 0 X X X Metals 8 Vitamin 0 X X X Mix

Recipe optimization used a matrix of parameter variability (Table 2: P1to P8) to define eight variable parameters and v0 to v5 represent testedvalues of each parameter. Each tube was composed using the standardvalues of each parameter (highlighted) except for one which was varied.In this round of sand pack experiments included 23 tubes plus blanks(all in triplicates).

Initial methanogen cultivation tests of production water from the Belugagas field revealed that Methanolobus had a salt optimum of 16 g/literwhen grown on methanol and trimethylamine (TMA). Nevertheless, when thesame production water was tested for salt optima using endemic coal as asubstrate, the salt optimum was found to be 4 g/liter. This resultrevealed that the salt requirements of the entire degradative pathwaymust be considered when designing consensus nutrient mixes.

Example 3 Stimulation of Methanogenic Degradation of Coal andRecalcitrant Organic Matter in Model Sandpack Bioreactors by AddingMethanolobus

Methanolobus taylorii was added to sand pack tubes amended with target(Cook Inlet) coal/organic debris/volcanic ash, and optimized nutrientadditions suspended in filter-sterilized, target production water. Ratesof methane production were stimulated with addition of M. taylorii (FIG.11).

Model sand pack bioreactors were set up as described in Example 2.Briefly, carbonaceous material (0.67 g each of coal, sandstone withorganic debris, and volcanic ash) from Cook Inlet core samples weremixed in the natural in situ ratios; with 17.8 g of sand (U.S. SilicaCompany, ASTM grade) in Hungate tubes (18×150 mm) fitted with blue,butyl rubber stoppers. The components had been transferred into ananaerobic chamber, which is where assembly of the tubes and inoculationswith microorganisms took place. The tubes containing carbonaceousmaterial/sand were overlayed with pure sand, stoppered and crimped withaluminum caps, and then autoclaved for 20 minutes at 120° C.

Sterile sand pack tubes, were brought back into the anaerobic chamber.Standard optimized, nutrient conditions were used (see Example 2) andthe final solutions, which also contained gas field production wateramended with endogenous microorganisms +/−M. taylorii, were introducedinto the tubes using a 20 gauge six inch spinal needle.

Methanolobus tayloriii (#9005) was purchased from the Deutsche Sammlungvon Mikrqorganismen und Zellkulturen GmbH (DSMZ™), Braunschweig, Germanyand grown in liquid culture using established techniques. The minimalessential medium (MEM-1) may be used as described by Zinder, S. H. In“Techniques in Microbial Ecology”. The MEM-1 was supplemented withtrimethylamine, 8 g/L sodium chloride; and mercaptoethane sulfonic acid(Coenzyme M), as well as the following antibiotics: vancomycin,streptomycin, kanamycin, penicillin G. M. taylorii was grown with anitrogen headspace at room temperature, shaking, until turbid. Frozenstocks (−80° C.) of the organism were made by mixing turbid cultureswith a 2× stock solution of freezing medium (final concentration 20%glycerol in MEM-1). For the sand pack inoculations, a concentratedfrozen stock (1.8 mL) Was thawed, added to reduced anaerobic mineralmedium (RAMM), and washed once to remove glycerol and antibiotics. Thecells were resuspended in production water plus nutrients beforeinoculation into sand pack tubes. The amount of cells added wasindeterminate.

All experimental conditions were performed in triplicate. Afterinoculation, the atmosphere in the headspaces of all tubes was replacedwith nitrogen, and the tubes were 5 incubated at 20° C.

Example 4 Synthetic Consortia of the Invention

This example describes an exemplary method of making a composition ofthe invention, a synthetic consortia, and e.g., the so-called“Consort-ABS1” composition of the invention.

A collection of production water samples from a biogenic gas reservoir(Cook inlet) was profiled and analyzed to test whether two-dimensionalcluster analysis of 16S rRNA gene sequences would reveal the presence ofa consortium of sequences (where the sequences serve as a proxy for thecorresponding microbe) whose abundance distribution among the samples asa group corresponded to methanogenesis activity.

To isolate total genomic DNA, production water samples (250-500 mls)were filtered through a 47 mm 0.2 pore size Durapore membrane filter(Millipore, Billerica, Mass.). Using a sterile scalpel, filters weresliced into 96 equal sized portions and transferred equally into two 2.0ml screw cap centrifuge tubes containing ceramic beads obtained fromCeroGlass (Columbia, Tenn.). The bead-beating matrix consisted of one4-mm glass bead (GSM-40), 0.75 g 1.4- to 1.6-mm zirconium silicate beads(SLZ-15), and 1.0 g 0.07- to 0.125-mm zirconium silicate beads (BSLZ-1)in 1 nil phosphate buffer (180 mM sodium phosphate, 18 mM EDTA, pH 8.0).Cells were disrupted in a Fastprep FP120 instrument as previouslydescribed (Ashby, Rine et al. 2007). Total genomic DNA was purified bycentrifuging the lysed cells at 13,200×g for 5 min at 4° C. Thesupernatants were transferred to 1.5 ml centrifuge tubes and 250 μl of2M potassium acetate pH 5.3 was added. The tubes were mixed by rotatingend-over-end and were centrifuged as above. The resulting genomic DNAwas purified on QIAprep Plasmid Spin columns (Qiagen, Valencia, Calif.)according to the manufacturer's instructions.

A portion of the 16S rRNA gene was amplified using the TX9/1391 primersas previously described (Ashby, Rine et al. 2007). Amplicons wereagarose gel purified and quantitated using SYBR green (Invitrogen,Carlsbad, Calif.). A second round of PCR was performed using fusionprimers that incorporated the ‘A’ and ‘B’ 454 pyrosequencing adaptersonto the 5′ ends of the TX9/1391 primers, respectively. The forwardfusion primer also included variable length barcodes that enabledmultiplexing multiple samples into, a single 454 sequencing run. Theseamplicons were PAGE purified and quantitated prior to combining into onecomposite library. The resulting library was sequenced using thestandard 454 Life Sciences Lib-L emulsion PCR protocol and Titaniumchemistry sequencing (Margulies, Egholm et al. 2005). Sequences thatpassed the instrument QC filters were also subjected to additionalfilters that required all bases be Q20 or higher and the average of allbases in any read to be Q25 or greater. Furthermore, the TX9 primer wastrimmed off of the 5′ end and the sequences were trimmed on the 3′ endat a conserved site distal to the V6 region (ca position 1067, E. colinumbering). The final sequences were approximately 250 bp in length andincluded the V5 and V6 regions.

The sequence abundance data was log transformed and clustered usingPearson correlation as the distance metric and Ward's method forhierarchical clustering. The clustering was performed using the softwareprogram PC-ORD. Inspection of the data revealed the organization ofsequences into, groups with one particular group showing a strongassociation with biogenic gas samples (FIG. 23). This presumptiveconsortium was comprised of 12 distinct sequences derived from threegenera including Acetobacterium, Bacteroidetes and Spirochaetes. Thisconsortium was labeled Consort-ABS1. The 12 sequences (the so-called“Consort-ABS1”) are:

SEQ ID NO: 1, AcetobacteriumCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAGACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCTGAGAGATCAGACTTTCCCTTCGGGGACAGAGAGACAGGT GGTGCSEQ ID NO: 2, AcetobacteriumCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAGACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCAAGAGATTGGGCTTTCCCTTCGGGGACAGAGAGACAGGT GGTGCSEQ ID NO: 3, AcetobacteriumCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAGACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCTGAGAGATCAGACTTTTCCTTCGGGAACAGAGAGACAGGT GGTGCSEQ ID NO: 4, AcetobacteriumCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAGACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCATCTGAGAGATCAGACTTTTCCTTCGGGAACAGAGAGACAGGT GGTGCSEQ ID NO: 5, AcetobacteriumCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAGACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCATCTGAGAGATCAGACTTTCCCTTCGGGGACAGAGAGACAGGT GGTGCSEQ ID NO: 6, AcetobacteriumCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAGACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCACCCAAGAGATTGGGCTTTCCCTTCGGGGACAGAGAGACAGGT GGTGCSEQ ID NO: 7, AcetobacteriumCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAGACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCAAGAGATTGGGCTTTCCCTTCGGGGACAGAGAGACAGGT GGTGCSEQ ID NO: 8, BacteroidesCACGCAGTAAACGATGATTACTAGCTGTTTGCGATACAATGTAAGCGGCTGAGCGAAAGCGTTAAGTAATCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTGAAATGCATCTGACCGGCCTTGAAAGAGGTTTTCCCTTCGGGGCAGATGTGTAGG TGCTGCSEQ ID NO: 9, SpirochaetesCGCACAGTAAACGATGTGCACCAGGTGGCGGGGGTAGAACCCCCGGTACCGTAGCAAACGCATTAAGTGCACCGCCTGGGGAGTATGCTCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGGTACGCGAGAAACCTTACCAGGGCTTGACATACACCGGAAGCGCCGTGAAAGCGGCGTGCCGCTTGCGGCCGGTGAAC AGGTGCTGCSEQ ID NO: 10, BacteroidesCACACAGTAAACGATGAATACTCGCTGTTTGCGATATACAGTAAGCGGCCAAGCGAAAGCATTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTGAATTGCAGAGGAACATAGTTGAAAGATTATGGCCGCAAGGTCTCTGTGAAGGT GCTGCSEQ ID NO: 11, BacteroidesCACACAGTAAACGATGAATACTCGCTGTTTGCGATATACAGTAAGCGGCCAAGCGAAAGCATTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTGAATTGCAGAGGAATATAGTTGAAAGATTATCGCCGCAAGGTCTCTGTGAAGGT GCTGCSEQ ID NO: 12, BacteroidesCACACAGTAAACGATGAATACTCGCTGTTTGCGATATACAGTAAGCGGCCAAGCGAAAGCATTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTGAATTGCAGAGGAATATAGTTGAAAGATTATAGCCGCAAGGTCTCTGTGAAGGT GCTGC.

To test whether Consort-A BS1 was capable of enhancing the rate ofconversion of coal to methane, the consortium was assembled from colonypurified isolates present in an in-house strain collection. The 12 16SrRNA gene sequences identified from the 454 sequence data that comprisedthe Consort-ABS1 consortium had 45 matches from the C600 straincollection with 100% identity and 100% coverage (Table 3). The strain IDnumbers comprised: 314, 316, 323, 325, 331, 339, 357, 362, 368, 372,386, 393, 462, 485, 557, 561, 571, 587, 591, 646.649, 650, 661, 662,669, 674, 675, 677, 679, 680, 682, 68.4, 686, 694, 696, 711, 712, 714,717, 722, 724, 726, 733, 734, 741. According to one aspect of theinvention, nucleic acid oligomers for the 16S rRNA gene, includingprimers with nucleotide sequences including SEQ ID NO. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12, may be used to identify strains in a consortium.

TABLE 3 Strain information from members of the Consort-ABS1 syntheticconsortium SEQ ID C600 Strain 454 V5V6 Genus NO 368, 372, 646TXv5v6-0101700 Acetobacterium 1 650, 674, 722 TXv5v6-0101090Acetobacterium 2 316, 331, 325, 362, TXv5v6-0101840 Acetobacterium 3537, 649 684, 714, 724 TXv5v6-0102484 Acetobacterium 4 675, 679, 680TXv5v6-0102389 Acetobacterium 5 650, 674, 722 TXv5v6-0102156Acetobacterium 6 339, 357, 650, 674, 722 TXv5v6-0101312 Acetobacterium 7677 TXv5v6-0028456 Bacteroidetes 8 314, 325, 557, 561, 571,TXv5v6-0005632 Bacteroidetes 12 587, 591, 662, 686, 694, 696, 712, 733,734 661, 682, 711, 714, 717, TXv5v6-0005526 Bacteroidetes 10 741 323,462, 669, 726 TXv5v6-0005680 Bacteroidetes 11 485, 393, 386TXv5v6-0239816 Spirochaetes 9

Each of these Strains was thawed from the strain library (stored at −80°C.), and patched out in an anaerobic chamber onto the following mediumplate types:

Strains 571, 587, 591, 717, 722, 724, 726, 733, 734, and 741 onAnaerobic Agar;

Strains 314, 316, 323, 325, 331, 339, 357, 362, 462, 485, 646, 649, 650,661, 662, 674, 675, 677, 679, 680, 682, 684, and 686 on Brucella BloodAgar Strains 368, 372, 386, 393, 537, 557, 561, 694, 696, 711, 712, and714 on Tryptic Soy Agarose.

After 14 days of growth on solid media, the patches were picked in ananaerobic chamber and transferred to liquid media: Anaerobic Broth,Brucella Broth, and Tryptic Soy Broth. After 20 days of growth, aConsortium mixture was prepared by pipetting 300 μl of each strain intoa sterile anaerobic conical, producing 9 ml of mixture. To this mix, 9ml of a 2× freezing medium was added: 2× Brucella Broth, 30% glycerol,0.05% sodium sulfide. The mix was then frozen at −80° C. for storage.

On the day of Sand Pack inoculation, 4 ml of the Consortium mixture wasthawed and then washed via centrifugation to remove any residualfreezing medium by centrifuging the mixture at 4° C., at 201×g for 20minutes followed by removing and discarding the supernatant. The pelletwas resuspended in 1 ml of production water by inverting several times.This preparation served as the inoculum for the Consort-ABS1 addition inthe sandpack experiment.

Sandpack tubes were set up to test the effect of a synthetic consortium(Consort-ABS1) on the coal to methane conversion rate in gas wellproduction water incubated with coal and other endogenous potentialsubsurface substrates comprising sandstone with organic debris andvolcanic ash. This experiment would in effect be supplementing thenative microbes present in the production water with the Consort-ABS1consortium. Since Consort-ABS1 did not contain a methanogen a source ofmethylotrophic methanogens (Methanolobus sp.) was included in theexperiment.

Approximately 17.8 g of U.S. Silica Company's ASTM graded sand was addedto a sterile 15 ml polypropylene conical tube (22.88 g was added to eachno carbon substrate controls). Approximately 0.167 g of each carbonsubstrate mixture (coal, volcanic ash, and sandstone with organicdebris) were weighed out and added to the sand in each conical tube. Tocreate the carbon substrate mixtures, coal, volcanic ash and organicdebris each of which had been obtained from four different Cook Inletcore samples were combined. Each conical tube was vortexed for 10 sec tohomogenize the carbon and sand mixture. Additional 3.5 g aliquots ofsand were weighed into weigh paper packets for each of the conicaltubes.

All conical tubes with the carbon and sand mixtures (as well as the allsand controls), the packets of sand, and 18×150 mm glass Balch tubes(Bellco No. 2048-00150) that were previously washed with Sparkleen 1,rinsed with deionized water and dried were brought into an anaerobicchamber filled with 5% H₂, and % CO₂, balanced with N₃. The caps to theconical tubes were unscrewed and loosely replaced in the chamber toallow gas exchange. These materials remained in the chamber for at leasttwo days before assembly.

To assemble the sand pack tubes, each carbon and sand mixture was gentlypoured into a test tube in the chamber and an aliquot of sand was addedto the top to create a top layer (approximately 1″ height) with nocarbon substrate. Each tube was capped with a rubber stopper, over whicha metal cap was crimped. The assembled tubes were autoclaved on fastexhaust for 20 minutes and immediately brought back into the anaerobicchamber.

Sand Pack2—Cell Additions:

To maintain the same concentration of nutrient additions between allcell addition treatments, 45.5 ml of the standard nutrient additionssolution described previously was mixed with 154.5 ml of sterile (0.2 umfiltered) production water (40-3) for a final volume of 200 ml. Thestandard nutrient mix was assembled such that the final concentrationsin the sandpack incubations would be: sodium phosphate, pH 7.5, 7.8 mM;NaCl—KCl, 100 mM; NH₄Cl, 5.6 mM; MgCl₂, 2 mM; and 1× trace metals andvitamin solution. Three sand pack tubes (with carbon substrates) were,inoculated as no cell blanks with 5.5 ml each of only this mixture usingthe same method previously described. The remaining 183.5 ml of theproduction water and nutrient mixture was inoculated with 1.22 ml ofcells (40-3) thawed anaerobically from frozen stock (previously storedat −80° C.) for a 150× cell dilution. Three sand pack tubes with carbonsubstrates and three tubes with only sand (no carbon) were inoculatedwith this production water with nutrients and endogenous cells (40-3).

The remaining inoculated production water with nutrients was split intoseven aliquots of 17.5 ml each in sterile 50 ml conical tubes.Additional cells from different sources were added to each conical tubeto create the various cell addition treatment inocula. To remove anyfreezing media, frozen stocks (−80° C.) of cell addition types were spunat 3000×g for 20 min. The supernatant was removed; and the cells wereanaerobically resuspended in 1 ml of sterile (0.2 um filtered)production water (40-3). Cell additions grown in antibiotics were alsowashed and anaerobically resuspended in sterile production water. A 250μl aliquot of the resuspended cells were removed and loaded into a 96well plate. OD readings of each cell addition type (including theendogenous cells, 40-3) were taken at 550 nm on a Biotek Epoch platereader. The volume of cells to add to each 17.5 ml aliquot wasdetermined based on their OD reading using the following formula:

$\frac{2*{{OD}\left( {21\text{-}6} \right)}*0.12\mspace{14mu}{ml}}{{OD}\left( {{cell}\mspace{14mu}{addition}} \right)}$

where 0.12 ml is the volume of the endogenous cells added per 17.5 mlaliquot (150× dilution). Based on the OD readings, the following volumesof resuspended cells were added to each 17.5 ml treatment aliquot, andinverted to mix:

‘No Cell Additions’: None ‘Methanogens Only’: 0.14 ml Mgen1 and 0.23 mlMgen2 ‘Consort-ABS1 + Methanogen’ 0.7 ml Consort-ABS1 and 0.14 ml Mgen1and 0.23 Mgen2

Three sand pack tubes (with carbon substrates) were inoculated for eachcell addition treatment using a venting method. In an anaerobic chamber,the tubes were opened and a 6″ sterile spinal needle was gently pushedthrough the sand mixture to the bottom with its plug inside. The plugwas removed, leaving the needle in the sand as a vent and 5.5 ml of thecorresponding cell addition inocula mixture was added to the top of thesand with a 5 ml serological pipet. The liquid inocula slowly saturatedthe sand mixture from the top until it reached the bottom. Oncesaturated, the needle was slowly removed, leaving a thin layer of liquidat the top of the sand The same needle was used for each replicatewithin a cell treatment. Once inoculated, the tubes were capped andcrimped, the headspaces were replaced, and they were stored at 20° C. aspreviously described.

Description of Cell Additions

I. 40-3 are cells that are endogenous to the production water used inthe sand pack experiments. The cells were isolated by centrifuging theproduction water, anaerobically, at 3000×g for 22 minutes at 4° C. Thesupernatant was removed and the cells were resuspended in standardfreezing medium containing Brucella broth, 0.05% sodium sulfide, and 15%glycerol. They were then stored at (−80° C.) until needed.

II. Mgen1 is an enrichment culture that was cultured from productionwater from Beluga well (40-6). The previously frozen cells wereinoculated into methanogen enrichment medium, MEM+trimethylamine+16 g/LNaCl, sodium sulfide, and antibiotics (vancomycin, streptomycin,kanamycin, penicillin G) and incubated for several months before it wasdetermined that the culture was making methane. It has been sequencedusing 454 sequencing technology and shown to contain a high proportionof Methanolobus. It was frozen until needed.

III. Mgen2 is a highly purified methanogen culture (that contains morethan one species) derived from production water 40-3. The previouslyfrozen cells from this Beluga well were inoculated intoMEM+peptone+sodium acetate+methanol+trimethylamine+sodiumformate+antibiotics (kanamycin, vancomycin, ampicillin)+sodium sulfide,and the culture in the serum vial was given a hydrogen overpressure.After a month the culture was shown to produce methane. An aliquot wasthen placed in a “Gellan Roll Vial” in order to isolate single colonics.The composition of the gellan roll vial was the same as the liquidculture except that no antibiotics were used and the gelling agent addedwas 0.7% gellan. A single colony was plucked from the vial afterincubation for one month and it was resuspended in liquid medium of thesame composition as described above. After two weeks this highlypurified culture was shown to produce methane. This culture was useddirectly in the sand packs.

IV. Consort-ABS1: 12 sequences were identified from the 454 sequencedata as clustering across multiple samples from biogenic gas wells whensorted by abundance, and were therefore selected as candidates for aConsortium mixture.

At various time points during incubation of the sand pack tubes, aportion of the headspace was removed to determine the amount of gasproduced using a pressure transducer. The amount of methane produced wasdetermined by gas chromatography analysis of headspace samples includinga correction for the total volume of gas produced. The rate ofconversion of coal to methane began to increase at 42 days in theConsort-ABS1 plus Methanogen and the No Cell Addition sand packincubations (FIG. 24). Interestingly, incubations supplemented withMethanogens alone appeared to detract from the methanogenic rate. Thehighest coal to methane rates from any of the conditions tested wereobserved in the Consort-ABS1 plus Methanogens from 70 days onward andthese differences were statistically significant.

Example 5 Creating Injectates Comprised of Microbes Capable of EnhancedMethanogenic Degradation of Organic Substrates

Microbial consortia derived from target (Cook Inlet) production waterwere selectively enriched with specific chemical compounds which areanalogues to target subsurface organic matter (FIG. 10). This methodallows relatively rapid growth of biomass in microbial consortia thatcould be re-injected, e.g. with the optimized nutrient mix. Thecoal/lignin-degrading consortium was derived, from target gas fieldproduction water, and is composed of microorganisms that were enrichedon lignin (SIGMA-ALDRICH), lignin monomers, and Cook Inlet gas fieldcoal. The lignin monomers that were tested were: ferulic acid, tannicacid, quinic acid, and shikimic acid, which are representative compoundstypical to low maturity coals, e.g. Beluga and Sterling formationorganic matter thermal maturity averages at 0.33% of R₀.

To obtain the consortia used for inoculation into sand pack tubes,enrichment cultures were established previously, assayed for pressureand methane production, passaged into medium of the same composition,and then frozen at −80° C. To prepare the parent cultures the lignin andlignin-like compounds were added to RAMM-Fresh medium (Shelton andTiedje 1984) along with a mixture of coals. Final concentration for alllignin and lignin-like compounds combined was 50 mg/L. The seven rocktypes, originating from target basins were ground together using amortar and pestle, and 0.9 g of the mixture was allocated anaerobicallyinto each vial of RAMM-F. Production water (3-4 mL) from target sites,which had been kept anaerobic, was inoculated into the vials, and anitrogen headspace was provided. The vials were incubated in the dark,shaking, at room temperature for several months. During the incubationthe headspace gases were assayed and the production water-consortia thatproduced high volumes of methane gas were noted. These corresponded tothe same production waters used in the sand pack experiments describedherein. Aliquots of these cultures were inoculated into fresh medium ofthe same composition to obtain P1 cultures. These cultures were thenincubated and monitored as described above. Frozen aliquots of theparent (P0) and passaged (P1) cultures were obtained by mixing theliquid cultures with 2× freezing medium (30% glycerol, 2× RAMM-F). Thecoal-degrading consortia used in the sand pack tubes was a frozen P1aliquot (2 mL) that was thawed, and then added to RAMM-F medium, washedonce, and resuspended in production water. The coal from the originalcoal/lignin enrichment was not removed. The amount of cells added wasindeterminate.

Target Basin Production Water

Target basin (California) production water was collected and keptanaerobic until used for sand pack experiments. The sand pack tubes wereassembled exactly as described in Example 3 except that for theseexperiments, California production water and endogenous 10 microbes wereamended with standard optimized nutrients at the concentrationsdescribed in Table 2. Cook Inlet coal/volcanic ash/sandstone withorganic debris was used in these sand pack tubes. The coal-ligninconsortium derived from Cook Inlet 40-5 production water, as describedin Example 4, was added to enhance methanogenesis rate (FIG. 10 b).

Identification of methanol-utilizing methanogenesis or “methylotrophic”conversion, provided new routes to increased biogenic gas production.Identification of the dominant gas production pathway given thecombination of microbial organisms, hydrocarbon substrate, and formationwater chemistry allows for increased biogas production rates, betterutilization of reservoir hydrocarbons, greater overall biogenic gasproduction and a longer life for biogenic gas reservoirs.

Example 6 Biogasification Risk Analysis

Applying a biogasification risk analysis process in the field (e.g.bioplugging and excess biomass development, oxygen-driven microbialcorrosion in injection lines, bio-sludge development in injection tanks,inorganic scale formation, field-wide redox status ofproduction/injection water, etc), was proposed in order to identifyproblems that could arise during field operations. A scheme forinjection of the optimized nutrient recipe was also used to identifysome of the potential problems (FIG. 12). The scheme is made up of 2separate tanks (A and B). Tank A is to be used for mixing concentratedstock solution of the optimized nutrient recipe, while tank B is to beused for storing nutrient solution received from tank A. The scheme alsoincludes ports for sample removal, and injection pumps to control flowof solutions exiting both tanks. According to the scheme, nutrientsolution exiting tank B is to be mixed with injection water atappropriate ratio in order to achieve the desired concentration ofnutrient components in the final mixture that is to be injected into thereservoir. Further, understanding of the travel length and thecorresponding travel time of solution from the point of mixture to thepoint of entry into the reservoir helped clarify the likelihood ofexcess biomass formation, bioplugging, and oxygen-driven bio-corrosionalong the injection line and in the well-bore. The potential forbiological and inorganic sludge formation and accumulation in the mixingand storage tanks was evaluated. The tests carried out included chemicalanalysis of reagent samples, field-wide redox measurement; biomasscontrol with sodium hypochlorite (NaOCl) solution; sludge treatment; aswell as scale formation and modeling.

The purity of chemical reagents to be used for developing solution ofnutrient recipe was confirmed by analyzing test samples of reagents thatwas obtained from selected Commercial vendors. The analytical testscarried out include ion chromatography (IC), inductively coupled plasmamass spectrometry (ICP-MS), and inductively coupled plasma atomicemission spectroscopy (ICP-AES). The overall objective of the analysisis to determine the level of purity of the reagents, as well as identifylevel of certain elements (e.g. sulfur) in the reagents which may posepotential problems if introduced into the reservoir. This allowsidentification of the appropriate vendor supplying purer forms of therequired chemical reagents (FIG. 18).

The procedures to test the effectiveness, and the minimum dose, of NaOClthat is required to eliminate biomass in the injection line andinjection well-bore during the field biogasification process are thefollowing. The initial population of microbes in production water,placed in tubes that were to be incubated under defined oxygensaturation conditions was determined either by direct counting of cellsusing a Petroff Hausser bacterial counting chamber, or by counting thecolony forming units (CFU/mL) of microorganisms that developed onspecialized media after incubation for specific period. The populationof microbes that were present in the incubated tubes (also with addedcarbon source-coal and nutrient additions) was determined over timeafter methane was detected in the headspace of the tubes. Thereafter,the same tubes were amended with different concentrations (0.6%, 0.3%,0.15%, 0.1%, 0.075%, 0.06%, 0.03% and 0%) of NaOCl solution followed byincubation of the tubes for 23 hours to allow the biomolecule-oxidizingaction of the solution to come to appreciable completion. Immediatelyafter this, the population of viable microbes (i.e. CFU/mL) in theNaOCl-amended tubes was determined by plating a specific volume of theculture on defined solid medium. All the concentrations of NaOClsolution that was tested were effective in inactivating microorganismsin the nutrient solution irrespective of the presence or absence ofoxygen (FIG. 19).

The potential for biomass production and accumulation in nutrientsolution in the mixing tank, storage tanks, and injection line wasevaluated as follows. Production water was amended with specificconcentrations of specially selected nutrients. To mimic theconcentration to be used for the storage tanks, the production water wasamended with excess concentration (i.e. 25×) of nutrients, whilenutrients amended into production water at lower concentrations (1×)mimic, the concentration of nutrients in solution entering the injectionline. The nutrient-amended water was transferred into test tubes, andthe initial population of microbes in the water in these tubes wasdetermined. Thereafter, the tubes were incubated at 10, 20, or 25° C.for different periods (9 and 30 days for 1× concentration; 7, 16, and 28and 50 days for 25× concentration) to allow growth of microbes anddevelopment of appropriate amount of biomass in the tubes if any. Thepopulation of microbes that developed after incubation was determinedusing the direct counting procedure (i.e. CFU/mL). Biomass levelsdropped after 50 days of incubation in the concentrated 25× nutrientsolution (FIG. 20.) although there was an initial increase in biomasslevel after seven days. Biomass level in the 1× nutrients recipeincreased over time (0-30 days), suggesting that the nutrient solutiononce in the injection line and injection well-bore may support thedevelopment of biomass in the zones.

The effectiveness of any one or combination of compounds with an imineor quinone functional group to remove oxygen from oxygen-exposedproduction water was tested. The inline solutions may contain one ormore imine oxygen scavengers including hydrazines, methylimines,ethylimines, propylimines, butylimines, diethylhydroxylamine (DEHA),alkeneimines like hydroxyalkylhydroxylamine, phenylenediamines,aminoguanidine, carbohydrazide, and the like. The quinone solutions maycontain one or more quinone oxygen scavengers including hydroquinone,orthoquinone, semi quinone, pyrroloquinoline-quinone (PQQ),methylhydroquinone and the like. Other non-sulfur containing oxygenscavengers may also be used like aldehydes, carboxylic acids like aceticacid and tartronic acid, carbohydroxide, erythorbate, cobalts,methylethylketoxime (MEKO) and the like. The extent of oxygen removalwas determined by measuring the change in redox potential ofnutrient-amended (i.e. 1× concentration) production water that waspreviously exposed to atmospheric oxygen. The concentration of theoxygen scavenging compounds that was tested is shown in

TABLE 4 The results from this test show that the compounds wereeffective in reducing oxygen saturation level in the produced water(FIG. 21A). Table 4: Composition of produced water amended with twooxygen-scavenging compounds Conditions A B C D E F G Composition^(a)Water Water, Water, Water, Water, Water, Water, and nutrients,nutrients, nutrients, nutrients, nutrients, nutrients, nutrients 34 mgof 34 mg of 68 mg of 136 mg 170 mg 204 mg imine imine + imine + ofimine + of X + of imine + 1.9 mg 3.8 mg 7.4 mg 9.3 mg 11.2 mg of of ofof of quinone quinone quinone quinone quinone ^(a)Final concentration ofimine and quinone in 1 L of aqueous nutrient solution.

Field measurement of redox potential (ORP) and oxygen saturation level,in produced water and injection water, determined if ORP and oxygensaturation level in the produced water and injection water vary in waterobtained from wells or facilities across the field. Measurement was doneusing a YSI 6920 V2 sonde (YSI, Ohio, USA) which also allowedsimultaneous evaluation of multiple parameters in water samples thatwere collected from the well-heads or water storage tanks directly.According to the information generated during the field sampling the ORPand oxygen saturation level vary across the field however watercollected directly from the well-heads exhibits lower ORP and oxygenlevel in comparison to water collected from storage tanks receivingwater from the respective wells. Water collected from vacuum trucks thatare transferring produced water from producing wells to the injectionwell in the field had the highest ORP and oxygen level (FIG. 22 B).

The effectiveness of defined concentration of chemical reagents (NaOCland acid) to act individually or complimentarily in dissolving sludgematerial that was collected from an on-site storage tank was determinedby adding 10 mL of 6% NaOCl solution to ˜2-3 g of sludge sample. Thiswas then mixed by a vortex mixer for 30 sec. The mixture was thenincubated at room temperature for 10, 30 or 60 min. Thereafter, themixture was filtered through a pre-washed (using 10 mL of 6% NaOClsolution followed by 10 mL deionized water) and pre-weighed 50 μmfilter. Then, 10 mL of deonized water was used to wash excess NaOClsolution. The filter and any residue that was left was then weighed.Alternatively, the filter with residue was exposed to 10 mL of 2N HClfor 10 minutes before filtration and washing with 10 mL deionized water.In all cases the amount of residue left on the filter after treatmentswas compared to the amount prior to treatment and the percent differencein weight was estimated (Table 5). Results show that NaOCl solution wasvery effective in dissolving sludge material and that treatment withacid (HCl solution) alone was not as effective. However, treatment ofsludge with NaOCl prior to treatment with acid increased dissolution ofthe sludge materials, most likely due to increased accessibility of theinorganic particles that are trapped within the sludge to acid afterNaOCl treatment.

TABLE 5 Effect of NaOCl solution and acid treatment on dissolution oftank derived sludge materials After NaOCl Total Incubation time SludgeWeight After NaOCl and HCl dissolved (min) (g) (Δg) (Δg) (%) 10 2.520.331 0.293 88.4 30 2.26 0.174 0.166 92.6 60 2.80 0.120 0.107 96.2

Finally, the tendency for inorganic scale formation in a solution thatcontained specific volume of produced water and defined amount ofnutrient recipe was determined by a combination of bench tests andchemical modeling of scale, formation. The procedures include:thermodynamic prediction modeling using SCALECHEM ™3.1 (OLI Systems) inorder to calculate scale formation by the mixtures; analysis of solidfiltrate collected by passing produced water through a 0.45 HV filter,and in which the solids were vacuum dried, weighed and subjected to FTIRand XRD/XRF in order to identify its composition; bottle tests usingfiltered (0.22 μm) produced water and nutrient recipe in which the majorconstituents in the recipe were added into the produced water followedby manual swirling of the mixture followed by incubation at stationaryposition for appropriate period and further analysis by inductivelycoupled plasma (ICP); bottle test at 80° F. by filtration of producedwater with 0.45 μm and 0.22 μm filters, and, thereafter, ammoniumphosphates and vitamin solution were equilibrated at 80° F. and thenadded into the produced water that was also equilibrated at a similartemperature to achieve the defined concentrations of nutrientcomponents. The mixtures were then incubated overnight on a shaker (85rpm) at 80° F. After that the mixtures were then filtered and analyzedby ICP in order to determine phosphate concentration in the solution;kinetics of generation of calcium phosphate solids at different initialcalcium concentration was determined by kinetic turbidity measurement.Then 125 μL of the different concentrated stock solutions of calcium wasadded individually into 2.5 mL of synthetic produced water-nutrientsolution placed in cuvettes. The absorbance of the mixtures was read at500 nm with a VARIAN CARY™ UV-Vis 1000 Spectrophotometer. In all cases,samples in the cuvettes were stirred during incubation at 47 and 80° F.

Thermodynamic modeling showed that there is the likelihood for inorganicscale formation in produced water-recipe mixture, suggesting that thefinal composition of optimized nutrient recipe is determined aftercareful consideration of the composition of the scale forming compoundsthat precipitates in the mixture. This method predicted the most likelyinorganic mineral scale based on the saturation index (SI) of theidentified compounds. Calcium-containing compounds were identified asmost likely inorganic mineral scale. In agreement with the result ofthermodynamic prediction, addition of nutrients into the produced waterled to depletion of calcium in the produced water-nutrient recipemixture as determined by ICP. Results also shows precipitation of somecalcium-containing compounds may cause depletion of other essentialnutrients in the produced water-nutrient recipe mixture.

Example 7 Reservoir Simulation of Biogas

Simulation of biogas in the sub-surface reservoir requires the abilityto model the flow of nutrient & microbe amended fluids and methanethrough porous media, as well as the ability to represent microbialgeneration of methane through chemical reactions. To accomplish thesetasks, computational modeling software was used, incorporating methanegeneration rates as derived from laboratory testing and a geocellularmodel which adequately represents the geologic variability inherent inthe reservoir. The generation of methane from microbial processes isrepresented through a series of chemical reactions involving thefollowing components: microbes, nutrients, and biodegradable coal volumein the sub-surface. Sub-surface microbe and nutrient volumes aredetermined from current conditions in the sub-surface, as analyzed fromproduced water samples; and assumed volumes of nutrient/microbeamendments to be injected into the sub-surface. Biodegradable coalvolume in the sub-surface reservoir may be calculated from petrophysicalinterpretation of coals observed in well logs and total organic carbon(TOC) measurements of sub-surface core samples for each lithology/and/orfacies expected to be contacted by injected fluids. Coal volume is thendiscounted based on the both the fraction of the coal that isbiodegradable and on the accessibility of these biodegradable coals tomicrobes in the sub-surface, e.g., lithologies or facies with lowerporosity or permeability will be difficult for microbes to move through,and, therefore, access the available organic matter, as compared tolithologies and/or facies with higher porosity/permeability (Table 6).The fraction of accessible biodegradable coal can then be populatedthroughout the gcocellular model based on the lithology/facicsdistribution previously defined for that model (FIG. 16).

TABLE 6 Coal volumes *Coal content Log- (TOC- Accessible Bio- basedbased) in Coal beds + coal Bio- degradable & Bio- coal, disseminateddisseminated fraction degradable accessible degradable Model Vol. form,Vol. coal, Vol. (based on coal coal coal Vol. Facies fraction fractionfraction Permeability perm.) fraction fraction % of rock Flood plain0.260 0.046 0.306 intermediate 0.50 0.250 0.125 3.829 to very lowAbandoned 0.100 0.046 0.146 intermediate 0.70 0.225 0.158 2.305 channelto low Crevasse 0.100 0.113 0.213 intermediate 0.75 0.225 0.169 3.600splay Channel 0.060 0.005 0.065 good 0.90 0.200 0.180 1.176 belt *forabandoned channel fraction, floodplain was used because the 2 coalyshale samples/intervals are likely included in the log-derived coals(these have very high TOC)

The sub-surface simulation of biogas as described above is highlydependent on understanding how applicable the methane generation ratesfrom laboratory testing are to the sub-surface. Given the large degreeof uncertainty in this understanding, it is desirable to test multiplemethane generation rates in order to understand the range of possiblemethane volumes generated and the travel time of methane generated nearthe injection well-bores to the producing and/or monitoring well-bores.In order to quickly test multiple scenarios, a simplified simulationapproach can be used to mimic the simulation described above, whilesignificantly decreasing the computer processing time required forsimulation. For this simulation, a range of potential biogas generationrates in the sub-surface are calculated by upscaling the biogasgeneration rates observed in laboratory experiments to the estimatedrock and fluid volumes expected in the sub-surface. These multiple ratesmay be further modified up or down by scaling factors to representpossible unknown conditions in the sub-surface and give a widervariation in potential outcomes. These various rates are thenrepresented as gas injection rates into the injection well-bore.Simulation of methane flow from injection well-bore toproducing/monitoring well-bores can then be done using standard flowsimulation software. Adding a small volume of a gas isotope tracer(described below) to the gas injection then allows the gas travel timefrom injection well-bore to producing/monitoring wellbore to be quicklyestimated for multiple biogas generation rates. The simulation resultsshown in FIG. 17 are based on an 18-month injection of gas at variousrates, representing the total volume of biogas expected, to be generatedduring the 18-month period. Once the tracer detection limit isestablished, the gas travel lime from injection wellbore toproducing/monitoring wellbore can be determined for the various biogasgeneration rates. This method can also be used to quickly test variousreservoir properties with uncertainties that may also affect the gasmovement through the reservoir. This will assist in identifyingreservoir properties which may need further investigation to narrow keyuncertainties and in determining the appropriate length of timenecessary for monitoring to detect newly generated biogas.

Example 8 Mimicking of Coal Monomer

Modeling microbial growth in a reservoir is difficult because thesubterranean carbon sources often have differing chemical compositions,limited surface area, microbial growth is restricted, and reactionconditions including pressures and temperatures are hard to replicate invitro. In order to quickly identify growth factors that improvemicrobial growth in situ, a method of growing microbial cultures invitro was required that both mimicked the subterranean formation andincreased surface area to allow for faster reaction times. Monomers wereidentified for various subterranean carbonaceous formations thatmimicked the chemical-bond structures present within, the targetedformation substrate. In addition to water environment, microbialassociations are likely to be partially controlled by the substratechemistry.

Chemical compositions that mimic substrate chemistry are readilyavailable and may be identified based on the structure and compositionof the carbon compounds in the reservoir. In some examples thesubstrates are selected from syringic acid; syringie acid methyl ester;dimethyl phenol; 2,4-dimethyl phenol; guaiacol; protocatechuic acid;vanillic acid; isovanillic acid; caffeic acid; ferulic acid; isoferulicacid; dibenzofuran; 8-amino 2-naphthanol; 7-methoxy coumarin; biphenyl4-methanol; 1,1′-biphenyl methyl; methoxy biphenyl; 3-methoxy biphenyl;dimethyl phenanthrene; dimethyl fluoranthene; 8,9-dimethyl fluoranthene;dimethylnaphthalene; dimethyl anthracene; acetylene; diacetylene;vinylacetylene; methyl naphthalene; trimethyl naphthalene;7-ethyl-1,4-dimethylazulene;trimer-3-methoxy-4-benzyloxy-alpha-(2-methoxyphenoxy)-b-hydroxypropiophenone;composition derived from the basic structure of lignin or kerogen; orother hydrocarbons found in the subterranean carbonaceous formation.Addition of these substrates to an aqueous culture, sand-packbioreactor, or as an additive to other growth media provides a method toidentify microorganisms that will preferentially degrade a carbonaceoussubstrate, and/or generate biogas from the carbonaceous substrate.

TABLE 7 Carbonaceous formation properties Vitrinite Reflectance SampleLocation(s) Rank (% R₀) Monomers Coal A Alaska Lignite/sub- 0.33 quinicacid, shikimic acid, bituminous C pannic acid, ferulic acid Coal B UtahHigh- 0.56 volatile bituminous C Coal C New Mexico High- 0.87 syringicacid; dimethyl phenol; volatile 8-amino 2-naphthanol; 7- bituminous Amethoxy coumarin; biphenyl 4- methanol; methoxy biphenyl; 1,1′-biphenylmethyl; dimethyl phenanthrene; dimethyl fluoranthene; and trimer-3-methoxy-4-benzyloxy-alpha-(2- methoxyphenoxy)-B- hydroxypropiophenoneCoal D New Mexico Medium- 1.10 syringic acid; dimethyl phenol; volatile8-amino 2-naphthanol; 7- bituminous methoxy coumarin; biphenyl 4-methanol; methoxy biphenyl; 1,1′-biphenyl methyl; dimethyl phenanthrene;dimethyl fluoranthene; and trimer-3- methoxy-4-benzyloxy-alpha-(2-methoxyphenoxy)-B- hydroxypropiophenone Peat Europe, Peat <0.3 humic,fibric, hemic, sapric North syringic, quinic, shikimic, America, pannic,and/or ferulic acids, as New well as compositions listed Zealand, belowAsia, Malaysia Coal Europe, Lignite ~0.25-0.38 Lignin carbonyl,carboxyl, North amidic, ester, phenolic, America, alcoholic, ketone,aldehyde, Asia, benzenoid, paraffinic, naphthenic Australia, andaromatic hydrocarbon India monomers Coal Wyoming Sub- ~0.38-0.6 bitumous

TABLE 7 Carbonaceous formation properties Vitrinite Reflectance SampleLocation(s) Rank (% R₀) Monomers Coal Brazil, High- ~0.5-1.1 Illinois,volatile Indiana bituminous Coal Medium- ~1.1-1.5 volatile bituminousCoal Low- ~1.5-1.9 volatile bituminous Coal Semi-  ~1.9-2.75 anthraciteCoal Anthracite ~2.75-6.0 

The discussion of any reference is not an admission that it is prior artto the present invention, especially any reference that may have apublication date after the priority date of this application. At thesame time, each and every claim below is hereby incorporated into thisdetailed description or specification as additional embodiments of thepresent invention.

Although the systems and processes described herein have been describedin detail, it should be understood that various changes, substitutions,and alterations can be made without departing from the spirit and scopeof the invention as defined by the following claims. Those skilled inthe art may be able to study the preferred embodiments and identifyother ways to practice the invention that are not exactly as describedherein. It is the intent of the inventors that variations andequivalents of the invention are within the scope of the claims whilethe description, abstract and drawings are not to be used to limit thescope of the invention. The invention is specifically intended to be asbroad as the claims below and their equivalents.

REFERENCES

All references, publications, patents, patent applications cited hereinare hereby expressly incorporated by reference for all purposes. Thediscussion of any reference is not an admission that it is prior art tothe present invention, especially any reference that may have apublication data after the priority date of this application.Incorporated references are listed again here for convenience:

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A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of making a synthetic consortiumcomprising: (i) obtaining two or more samples from at least twodifferent sources, wherein each sample comprises a plurality ofmicrobes; (ii) determining the microbial composition and abundance ofthe samples, wherein the abundance of each observed microbe is recordedfor each sample; (iii) performing a correlation analysis of abundancesof one microbe in the two or more samples to abundances of anothermicrobe in the two or more samples; (iv) repeating step (iii) for atleast one additional pair of microbes in said samples; (v) identifying agroup of two or more microbes, whose abundance correlation to oneanother is greater than to other microbes in the samples; and (vi)assembling a synthetic microbial consortium by physically combining atleast two microbial cultures of the identified microbes.
 2. The methodof claim 1, wherein at least one source comprises a carbonaceous sourceor formation.
 3. The method of claim 2, wherein the carbonaceous sourceor formation further comprise a coal formation, a peat, a lignite, abituminous coal, an anthracite coal, a coal analogue or precursor, aheavy oil, asphaltenes, or an organic debris.
 4. The method of claim 1,further comprising testing whether the synthetic microbial consortium isable to perform a specific function or process of interest in a cultureor in a sample.
 5. The method of claim 4, further comprising testingwhether the assembled synthetic consortium is able to convert acarbonaceous substrate into a lower molecular weight product, whereinthe method makes a synthetic consortium capable of converting acarbonaceous substrate into a lower molecular weight product byidentifying a synthetic consortium or a group of synthetic consortiathat can convert a carbonaceous substrate into a lower molecular weightproduct.
 6. The method of claim 1, wherein at least one of the twodifferent sources comprises a production water, a core, a cutting, anoutcrop sample, or an enrichment culture.
 7. The method of claim 1,wherein the microbial abundance for each microbe is log transformed. 8.The method of claim 1, wherein the identifying a group of two or moremicrobes comprises a hierarchical cluster analysis of a microbialdistribution across the samples.
 9. The method of claim 8, wherein thehierarchical cluster analysis is performed using a hierarchicalclustering algorithm, or a Ward's method.
 10. The method of claim 1,wherein the identifying a group of two or more microbes of the syntheticmicrobial consortium comprises at least one of the group consisting of:(a) a culture independent molecular survey, (b) deducing from theabundance data in a culture independent survey, or (c) using distancemetrics.
 11. The method of claim 10, wherein the culture independentmolecular survey comprises counting a number of copies of each distinct16S rRNA gene sequence detected from each sample.
 12. The method ofclaim 1, wherein the correlation analysis is selected from at least oneof the group consisting of: (a) Euclidean distance, (b) Chi square, (c)City block, or (d) an ordination method.
 13. The method of claim 12,wherein the ordination method is selected from at least one of the groupconsisting of: (a) Principal Components Analysis (PCA), (b) Bray-Curtis,or (c) Nonmetric multidimensional scaling.
 14. The method of claim 1,wherein determining the microbial composition and abundance furthercomprises sequencing of all or a portion of an rRNA gene.
 15. The methodof claim 1, wherein the abundance comprises a nucleic acid, DNA or RNArecorded for each sample.
 16. The method of claim 1, wherein thecorrelation analysis comprises a Pearson correlation.
 17. The method ofclaim 1, further comprising correlating the synthetic consortium to abiogeochemical process, or a metabolic intermediate or product.
 18. Themethod of claim 1, wherein the assembling the synthetic microbialconsortium further comprises cultivating the microbes using differentnutrient combinations to obtain single colonies.
 19. The method of claim18, wherein at least one of the medium formulations comprises acarbonaceous substrate.
 20. The method of claim 1, wherein assemblingthe synthetic microbial consortium further comprises cultivating themicrobes using anaerobic methods conditions.
 21. The method of claim 1wherein the microbial culture is selected from at least one of the groupconsisting of: (a) a pure culture, (b) a nearly pure culture, (c) anenrichment culture, or (d) a type strain.
 22. The method of claim 1,further comprising constructing a distance matrix.
 23. A method ofidentifying and making a synthetic microbial consortium comprising: (a)obtaining a plurality of samples comprising microbes; (b) determiningthe abundances of a plurality of microbes in the plurality of samples,wherein the abundance of each microbe is recorded for each sample; (c)compiling a numerical abundance of an environmental parameter of saidplurality of samples; (d) performing a correlation analysis of theabundance of one microbe in said plurality of samples to the abundanceof the environmental parameter in said plurality of samples, wherein thecorrelation analysis provides a comparison between the abundance of themicrobe to the abundance of the environmental parameter; (e) repeatingstep (d) for at least one additional microbe in said plurality samples;(f) identifying at least two microbes whose correlation to theenvironmental parameter is greater than other microbes in said pluralityof samples, (g) obtaining microbial cultures of at least two identifiedmicrobes; and (h) combining the microbial cultures of the identifiedmicrobes, thereby making a synthetic consortium.
 24. The method of claim23, wherein the environmental parameter comprises a biochemical process,or a metabolic intermediate or product.
 25. The method of claim 23,further comprising performing a hierarchical cluster analysis of thecorrelations.
 26. The method of claim 23 wherein the microbial cultureis selected from at least one of the group consisting of: (a) a pureculture, (b) a nearly pure culture, (c) an enrichment culture, or (d) atype strain.
 27. The method of claim 23, wherein at least one samplecomprises a carbonaceous source or formation.
 28. The method of claim27, wherein the carbonaceous source or formation further comprises acoal formation, a peat, a lignite, a bituminous coal, an anthracitecoal, a coal analogue or precursor, a heavy oil, asphaltenes, or anorganic debris.
 29. The method of claim 23, wherein at least one samplecomprises a production water, a core, a cutting, an outcrop sample, oran enrichment culture.
 30. The method of claim 23, wherein the abundancefor each microbe is log transformed.
 31. The method of claim 23, whereinthe identifying a group of two or more microbes of the syntheticconsortium comprises at least one of the group consisting of: (a) aculture independent molecular survey, (b) deducing from the abundancedata in a culture independent survey, or (c) using distance metrics. 32.The method of claim 31, wherein the culture independent molecular surveycomprises counting a number of copies of distinct 16S rRNA gene sequencedetected from each sample.
 33. The method of claim 23, wherein thecorrelation analysis is selected from at least one of the groupconsisting of: (a) Euclidean distance, (b) Chi square, (c) City block,or (d) an ordination method.
 34. The method of claim 33, wherein theordination method is selected from at least one of the group consistingof: (a) Principal Components Analysis, (b) Bray-Curtis, or (c) Nonmetricmultidimensional scaling.
 35. The method of claim 23, whereindetermining the abundances of a plurality of microbes further comprisessequencing of at least a portion of an rRNA gene.
 36. The method ofclaim 35, wherein the abundance comprises a nucleic acid, DNA or RNArecorded for each sample.
 37. The method of claim 23, wherein thecorrelation analysis comprises a Pearson correlation.
 38. The method ofclaim 23, wherein the obtaining microbial cultures of at least twoidentified microbes further comprises cultivating the microbes usingdifferent nutrient combinations to obtain single colonies.
 39. Themethod of claim 23, wherein the obtaining microbial cultures of at leasttwo identified microbes further comprises cultivating the microbes usinganaerobic methods.